Novel peptides and combination of peptides and scaffolds thereof for use in immunotherapy against colorectal carcinoma (crc) and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T-cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the continuation of U.S. application Ser. No.15/145,990, filed May 4, 2016, which claims priority to U.S. 62/157,684,filed May 6, 201 and GB1507719.1, filed May 6, 2015, the content ofwhich are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (seeM.P.E.P. § 2442.03(a)), a Sequence Listing in the form of anASCII-compliant text file (entitled“Sequence-Listing_2912919-046008_ST25.bd” created on 27 Sep. 2019, and42,059 bytes in size) is submitted concurrently with the instantapplication, and the entire contents of the Sequence Listing areincorporated herein by reference.

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT-cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is the third most common cancer in men and thesecond most common cancer in women. Globally, CRC accounts for about 10%of all newly diagnosed cancer cases. In 2012, 1.36 million new CRC caseswere diagnosed with 746,000 cases in men and 614,000 cases in women,resulting in a male:female ratio of 1.2:1 (World Cancer Report, 2014).CRC is a disease of the elderly. The mean age at the time of diagnosisis 68 years (SEER Stat facts, 2014).

Incidence rates vary geographically about ten-fold with similarities inmen and women. The highest incidence rates in both sexes occur inAustralia/New Zealand (age-standardized rate (ASR)=45 per 100,000 menand ASR=32 per 100,000 women). Incidence rates in Europe show smallregional variation and ASR=38 per 100,000 men and ASR=25 per 100,000women. The lowest incidence rates in the world occur in Western Africawith 4.5 per 100,000 men and 3.8 per 100,000 women (World Cancer Report,2014).

The overall 5-year survival rate from CRC is about 65%. However,survival rates depend on stage at the time point of diagnosis. The5-year survival for localized CRC is 89.8%, for regional and distant CRC70.5% and 12.9%, respectively. CRC is the fourth highest cause of cancerdeath (694,000 deaths; 8.5%) (SEER Stat facts, 2014; World CancerReport, 2014).

CRC is usually staged using the TNM system, which incorporatesinformation about the size of the primary tumor (T), the involvement oflymph nodes (N) and the occurrence of distant metastases (M). The UICC(Union Internationale Contre le Cancer) staging is based on the TNMsystem and includes statistical data for prognosis prediction(Stintzing, 2014).

Risk factors for developing CRC include lifestyle factors, hereditarydisposition and inflammatory conditions. Excessive alcohol use,cigarette smoking and obesity are associated with an elevated risk todevelop CRC. Hereditary risk factors are familial occurrence of CRC,familial adenomatous polyposis (FAP), attenuated FAP (AFAP)/attenuatedadenomatous polyposis coli (AAPC), hereditary non-polyposis colorectalcarcinoma (HNPCC) and hamartomatous polyposis syndromes. Inflammatoryconditions associated with an increased CRC risk include inflammatorybowel diseases (IBD) such as ulcerative colitis and Crohn's disease(Baena and Salinas, 2015; Stintzing, 2014; Vasen et al., 2015).

Histologically, more than 90% of all CRC are adenocarcinomas. Rare CRCtypes include neuroendocrine, squamous cell, adenosquamous, spindle celland undifferentiated carcinomas (Fleming et al., 2012). The majority ofcolorectal adenocarcinomas derive from adenoma or dysplasia precursorlesions. Depending on the type of the lesions/carcinomas, differentmolecular mechanisms contribute to tumorigenesis. The chromosomalinstability (CIN) pathway (“suppressor” pathway) is characterized bymutations in the APC, KRAS or p53 genes. Additional mutations are foundin the LKB1/STK11, SMAD4, BMPR1A or MYH genes. The microsatelliteinstability (MSI) pathway (“mutator” pathway) comprises mutations in theDNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, MMR genehypermethylation or BRAF mutations. Epigenetic instability, includingDNA methylation, histone alteration and chromatin remodeling, ischaracteristic for CIMP (CpG island mathylator phenotype) tumors(Fleming et al., 2012).

Depending on the CRC stage, different standard therapies are availablefor colon and rectal cancer. Standard procedures include surgery,radiation therapy, chemotherapy and targeted therapy for CRC (Berman etal., 2015a; Berman et al., 2015b).

Removal of the tumor is essential for the treatment of CRC. Anatomicconditions differ for rectal carcinomas from other CRC as the rectum islocated in the pelvis and the tumor can be difficult to access.Well-differentiated small rectal tumors (stage T1) require excision, butno further treatment with chemotherapy. Patients with rectal tumors ofhigher T stages receive neoadjuvant radio-chemotherapy with afluoropyrimidine prior to total mesorectal excision (TME) and adjuvantchemotherapy. For chemotherapeutic treatment the drugs capecitabine or5-fluorouracil (5-FU) are used. For combinational chemotherapy acocktail containing 5-FU, leucovorin and oxaliplatin (FOLFOX) isrecommended (Stintzing, 2014; Berman et al., 2015b).

Treatment of colon carcinomas involves radical hemicolectomy and lymphnode resection. Early stages (UICC stage I) do not require additionaltreatment. Patients with tumors of UICC stage II receive 5-FU orcapecitabine. Treatment for patients with UICC stage III includes thedrug combinations FOLFOX and XELOX (capecitabine plus oxaliplatin)(Berman et al., 2015a; Stintzing, 2014).

Metastatic, unresectable CRC are treated with chemotherapeuticalcocktails such as FOLFIRI (5-FU, leucovorin, irinotecan), FOLFOX,FOLFOXIRI (5-FU, irinotecan, oxaliplatin), FOLFOX/capecitabine,FOLFOX/oxaliplatin, FOLFIRI/capecitabine and irinotecan or UFT (5-FU,tegafur-uracil) (Stintzing, 2014).

In addition to chemotherapeutic drugs, several monoclonal antibodiestargeting the epidermal growth factor receptor (EGFR, cetuximab,panitumumab) or the vascular endothelial growth factor-A (VEGF-A,bevacizumab) are administered to patients with high stage disease. Forsecond-line and later treatment the inhibitor for VEGF aflibercept, thetyrosine kinase inhibitor regorafenib and the thymidylate-synthetaseinhibitor TAS-102 and the dUTPase inhibitor TAS-114 can be used(Stintzing, 2014; Wilson et al., 2014).

Latest clinical trials analyze active immunotherapy as a treatmentoption against CRC. Those strategies include the vaccination withpeptides from tumor-associated antigens (TAAs), whole tumor cells,dendritic cell (DC) vaccines and viral vectors (Koido et al., 2013).

Peptide vaccines have so far been directed against carcinoembryonicantigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognizedby T-cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG),Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, p53, ring fingerprotein 43 and translocase of the outer mitochondrial membrane 34(TOMM34), or mutated KRAS. In several phase I and II clinical trialspatients showed antigen-specific CTL responses or antibody production.In contrast to immunological responses, many patients did not benefitfrom peptide vaccines on the clinical level (Koido et al., 2013; Miyagiet al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprise DCs pulsed with either TAA-derivedpeptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA orDC-tumor cell fusion products. While many patients in phase I/II trialsshowed specific immunological responses, only the minority had aclinical benefit (Koido et al., 2013).

Whole tumor cell vaccines consist of autologous tumor cells modified tosecrete GM-CSF, modified by irradiation or virus-infected, irradiatedcells. Most patients showed no clinical benefit in several phase II/IIItrials (Koido et al., 2013).

Vaccinia virus or replication-defective avian poxvirus encoding CEA aswell as B7.1, ICAM-1 and LFA-3 have been used as vehicles in viralvector vaccines in phase I clinical trials. A different study usednonreplicating canarypox virus encoding CEA and B7.1. Besides theinduction of CEA-specific T-cell responses 40% of patients showedobjective clinical responses (Horig et al., 2000; Kaufman et al., 2008).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and CRC in particular. There is also aneed to identify factors representing biomarkers for cancer in generaland CRC in particular, leading to better diagnosis of cancer, assessmentof prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T-cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T-cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T-cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T-cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T-cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killerT-cells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T-cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T-cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T-cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T-cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T-cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T-cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T-cell can be found. Such afunctional T-cell is defined as a T-cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T-cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 191 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 191, wherein said variant binds to MHC and/or induces T-cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 191 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 191,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4A and B are furthermore useful inthe diagnosis and/or treatment of various other malignancies thatinvolve an over-expression or over-presentation of the respectiveunderlying polypeptide.

TABLE 1 Peptides according to the present invention SEQ ID No. SequenceGeneID(s) Official Gene Symbol(s)   1 ALIKQLFEA 168417, 441234,ZNF679, ZNF716, SAPCD2 89958   2 ALLPRYFFL 23120 ATP10B   3 RLIPDTLYSV1303 COL12A1   4 RLAELTVDEFL 26155, 401010 NOC2L, LOC401010   5WLFDDGGLTL 6557, 6558, SLC12A1, SLC12A2, 6559 SLC12A3   6 FLAELPGSLSL5326 PLAGL2   7 YLTRHLAVL 4583 MUC2   8 ALMLQGVDLL 3329 HSPD1   9ILDDHLSRV 8313 AXIN2  10 RMYNKIFAI 80201 HKDC1  11 YLFEKTFNM 90161HS65ST2  12 ALVQGILERV 4843 NOS2  13 FLLAEDTKV 10592 SMC2  14 FLDKPEDVLL2036 EPB41L1  15 LQLDKEFQL 24140 FTSJ1  16 VLVDQSWVL 5655 KLK10  17ALAAARVEL 6558 SLC12A2  18 FLSSLKGGLL 83608 C18orf21  19 RLYTKLLNEA 4651MYO10  20 YLKDGDVML 11180 WDR6  21 VLIDHRWVL 43849 KLK12  22 GLIDEVMVL54905 CYP2W1  23 FLDANGHFV 54905 CYP2W1  24 VLDGVLMEL 4190 MDH1  25SLADRLIGV 57535 KIAA1324  26 GLASKENFSNVSL 6840 SVIL  27 LLADEDSSYL51510 CHMP5  28 ALTEIQEFI 5591 PRKDC  29 QMLDVAIRV 8943 AP3D1  30GLSSAYGGL 10787, 3856, NCKAP1, KRT8, KRT8P3 728638  31 LLYGKYVSV 84065TMEM222  32 KLNTETFGV 149986 LSM14B  33 ALWEKNTHL 11190 CEP250  34ILLEKSVSV 80728 ARHGAP39  35 KLLDLTVRI 10562 OLFM4  36 GLLESPSIFNFTA23185 LARP4B  37 GLFAGLGGAGA 10916 MAGED2  38 SLAPTPVSA 8870 IER3  39GLNGGSPAAA 1045 CDX2  40 ALSNVIHKV 5268 SERPINB5  41 ILDDSFKLL 9843 HEPH 42 SILDDSFKL 9843 HEPH  43 TLDAAQPRV 6649 SOD3  44 SLESKLTSV 9289 GPR56 45 ALAELLHGA 26470 SEZ6L2  46 GLDDRYSLV 11187 PKP3  47 KLYERCEVV 2065ERBB3  48 FLDASDPAL 65266 WNK4  49 SGMGGITAV 3856 KRT8  50 TLMAEMHVV2186 BPTF  51 QVWEIQHTV 26290 GALNT8  52 ALDSSNSMQTI 3875 KRT18  53FLLGSEIKL 54885 TBC1D8B  54 ALLNGEYLLAA 57418 WDR18  55 QIITSVVSV 5378PMS1  56 VLFTDEGVPKFL 4731 NDUFV3  57 NLLEKENYL 5318 PKP2  58 AMADKMDMSL10189 ALYREF  59 LLTDNVVKL 79810 PTCD2  60 VLDEDEPRFL 23287 AGTPBP1  61KLLKLFQGV 26058 GIGYF2  62 YLAPENGYL 6625 SNRNP70  63 KLFSILSTV 54919HEATR2  64 KTLGKLWRL 30812, 6662, SOX8, SOX9, SOX10 6663  65 FGAPGIISA5725 PTBP1  66 GLDDGPDFL 58533 SNX6  67 SLNDLEKDVMLL 6597 SMARCA4  68SILQFVHMV 5800 PTPRO  69 GMLNEAEGKAIKL 4629 MYH11  70 MISELEVRL 4629MYH11  71 RLVVTEIPTAI 3710 ITPR3  72 YLLDYPNNLL 26057 ANKRD17  73YLFDIAVSM 51074 APIP  74 YLMGFLHAV 23779, 553158, ARHGAP8, PRR5-ARHGAP8,55615 PRR5  75 EMIENIQSV 1080 CFTR  76 YLIGEKQHYL 7429 VIL1  77SLLKRDFGA 1655 DDX5  78 ALDPELLLL 57805 KIAA1967  79 SLAADQLLKL 9295SRSF11  80 QVDEVVDIMRV 3604, 6844, TNFRSF9, VAMP2, VAMP3 9341  81ALLSQQTHL 7050 TGIF1  82 QLYEEPDTKL 10270 AKAP8  83 LTIEDGIFEV3306, 3312, HSPA2, HSPA8, HSPA8P8 100287551  84 SMVEDITGLRL 1832 DSP  85ILHDINSDGVL 4924 NUCB1  86 KVFPGKISV 56667 MUC13  87 LLFDAPDLRL 55561CDC42BPG  88 KLDIKVETV 55243 KIRREL  89 SLIEYEFRV 3655 ITGA6  90GLLKPGLNVVL 10969 EBNA1BP2  91 TVDVATPSV 8924 HERC2  92 WIDDTSAFV 5073PARN  93 SLQELRLLL 55502 HES6  94 KSMDIVLTV 4586, 727897 MUC5AC, MUC5B 95 AILDAHIEV 26290 GALNT8  96 KLYSRLVYV 387496 RASL11A  97 ALWWGVVTV3784 KCNQ1  98 AMNGKSFSV 79572 ATP13A3  99 KLLEVDLDTV 4648 MYO7B 100SLDDFLATA 55341 LSG1 101 GLSEGHTFQV 2318 FLNC 102 KILVSLIEV 10422 UBAC1103 FLFGYPKRL 64110 MAGEF1 104 ILLTIKDDTIYL 4583 MUC2 105 YALDLSTFL 8870IER3 106 SLISEKILL 26504 CNNM4 107 ALLGGGPYML 80004 ESRP2 108SLAELVPGVGGI 9742 IFT140 109 ALDGDQMEL 3192 HNRNPU 110 LLGELPRLLLL 1604CD55 111 HMDDGGYSM 27316, 494115 RBMX, RBMXL1 112 KLGQVLIYL 51809 GALNT7113 ILYDLQQNL 3783 KCNN4 114 TAVGHALVL 1293 COL6A3 115 SLFDVSHML 275 AMT116 LVYQFVHPI 25803 SPDEF 117 TLQPVDNSTISL 1266 CNN3 118 LLADLKTMV5141, 5142, PDE4A, PDE4B, PDE4C, 5143, 5144 PDE4D 119 ILYQTVTGL 83732RIOK1 120 VLYEGVDEV 93432 MGAM2 121 SLAPNIISQL 25824 PRDX5 122 SLMGMVLKL11169 WDHD1

TABLE 2 Additional peptides according to the present invention with noprior known cancer association-J = phosphoserine SEQ ID No. SequenceGeneID(s) Official Gene Symbol(s) 123 KTLERSYLL 6240 RRM1 124RVLPPSALQSV 9212 AURKB 125 KLGDFGLLVEL 9088 PKMYT1 126 TLAKYLMEL891, 9133 CCNB1, CCNB2 127 RLAELTVDEFLA 26155 NOC2L 128 MLDDRAYLV 23511NUP188 129 VLIDVLKEL 23019 CNOT1 130 GLGGSQLIDTHL 23215 PRRC2C 131KLLDVVHPA 10574 CCT7 132 ALLNAILHSA 25926 NOL11 133 RTFEKIEEV 3978 LIG1134 GVAGGSILKGV 1968, 255308 EIF253, LOC255308 135 KLQEEIPVL 1062 CENPE136 KLFDIFSQQV 55737 VPS35 137 QLTEIKPLL 57446 NDRG3 138 KQFEGTVEI 675BRCA2 139 VLLNEILEQV 64151 NCAPG 140 LLNEILEQV 64151 NCAPG 141 AVIEHLERL283459 GATC 142 SLVQRVETI 1894 ECT2 143 KLSDVWKEL 197259 MLKL 144LLNDRIWLA 90204 ZSWIM1 145 LLLEVVKQV 65065 NBEAL1 146 ALSDETWGL 2886GRB7 147 TLTELRAFL 8242 KDM5C 148 RLLENMTEVV 23042 PDXDC1 149YQFDKVGILTL 8563 THOC5 150 RLADLEALKV 10535 RNASEH2A 151 SAQGSDVSLTACKV100507703, LOC100507703, HLA-A 3105 152 KLLAVIHEL 25788 RAD54B 153ILFSEDSTKLFV 84916 CIRH1A 154 KLPSETIFVGC 50628 GEMIN4 155 RLLGEEVVRV9894 TELO2 156 SLMMTIINL 7153 TOP2A 157 SLIERDLKL 9875 URB1 158GLLDPSVFHV 79050 NOC4L 159 VLVDDDGIKVV 79022 TMEM106C 160 KLLEFDQLQL8871 SYNJ2 161 FLKNELDNV 10293 TRAIP 162 KLMDYIDEL 85444 LRRCC1 163RLLHEVQEL 10540 DCTN2 164 KMLDEILLQL 5425 POLD2 165 RLLDFPEAMVL 23113CUL9 166 GLLEARGILGL 990 CDC6 167 SVIDHIHLISV 10755 GIPC1 168 GLIRFPLMTI55643 BTBD2 169 YLAHFIEGL 64328 XPO4 170 ALAGGITMV 790 CAD 171RLQETEGMVAV 10042 HMGXB4 172 LLLDTVTMQV 22820 COPG1 173 KLGDLMVLL 57647DHX37 174 ILLDDNMQIRL 5261 PHKG2 175 TLLGGKEAQALGV 94059 LENG9 176RTLDKVLEV 9933 KIAA0020 177 ALLQGAIESV 25894 PLEKHG4 178 YLFREPATI 4728NDUFS8 179 RLLJPLSSA 125950 RAVER1 180 NLLEIAPHL 2820 GPD2 181NLFDLGGQYLRV 22827 PUF60 182 SLNKWIFTV 339665 SLC35E4 183 TLQEVVTGV55750 AGK 184 SLLDENNVSSYL 5591 PRKDC 185 VLYTGVVRV 64682 ANAPC1 186KMSEKILLL 5690 PSMB2 187 GLHNVVYGI 23019 CNOT1 188 FLVDGPRVQL 90204ZSWIM1 189 AISEVIGKITA 9183 ZW10 190 AMAEMVLQV 9918 NCAPD2 191 QLFSEIHNL55755 CDK5RAP2

TABLE 3 Peptides useful for e.g. personalized cancer therapies-J =phosphoserine SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 192KIQEMQHFL 4321 MMP12 193 KLSPTVVGL 8313 AXIN2 194 SLYKGLLSV 25788 RAD54B195 LLLGERVAL 23475 QPRT 196 KIQEILTQV 10643 IGF2BP3 197 SLFGQDVKAV26036 ZNF451 198 VLYGPDVPTI 4680 CEACAM6 199 FLLEREQLL 165055 CCDC138200 SAVDFIRTL 9263 STK17A 201 GJFNGALAAV 39 ACAT2 202 GLAALAVHL 2175FANCA 203 KLIDLSQVMYL 346389 MACC1 204 KLLDLETERILL 2803 GOLGA4 205RLHDENILL 23322 RPGRIP1L 206 RIAGIRGIQGV 23167 EFR3A 207 KLCEGFNEV51142, 646630 CHCHD2, CHCHD2P8 208 RLIDRIKTV 60560 NAA35 209 KLQDGLLHI7076 TIMP1 210 KLAVALLAA 3576 IL8 211 SLFGKKYIL 2274 FHL2 212 FLLDGSANV1293 COL6A3 213 LLWAPTAQA 389812 LCN15 214 SVLEKEIYSI 127602 DNAH14 215KLQEKIQEL 1062 CENPE 216 YLWDLDHGFAGV 832 CAPZB 217 KLLDTMVDTFL100527963, PMF1-BGLAP, PMF1 11243 218 KLSWDLIYL 51148 CERCAM 219FLDEKGRCV 4583 MUC2 220 KMDPVAYRV 5859 QARS 221 ILNVDGLIGV 47 ACLY 222GVIAEILRGV 10528 NOP56 223 VLMQDSRLYL 983 CDK1 224 QLQEGKNVIGL 8407TAGLN2 225 YLYGQTTTYL 7153 TOP2A 226 FLVDGSWSV 1303 COL12A1 227LTAPPEALLMV 79050 NOC4L 228 SMSGYDQVL 3187, 3188 HNRNPH1, HNRNPH2 229YLLEKFVAV 1663, 440081, DDX11, DDX12P, 642846 LOC642846 230 AMSSKFFLV7474 WNT5A 231 RLFADILNDV 64755 C16orf58 232 RLLDSVSRL 3918 LAMC2 233RLDDLKMTV 3918 LAMC2 234 KMFESFIESV 5576 PRKAR2A 235 LLHEENFSV 6942TCF20 236 KMSELQTYV 1063 CENPF 237 KLVEFDFLGA 10460 TACC3 238 NMLEAVHTI7272 TTK 239 QLIEKNWLL 56992 KIF15 240 VLAPRVLRA 5954 RCN1 241 ILIDWLVQV891 CCNB1 242 RLEEDDGDVAM 10482 NXF1 243 TLMDMRLSQV 24148 PRPF6 244SLHFLILYV 487, 488 ATP2A1, ATP2A2 245 QLIDYERQL 11072 DUSP14 246GLTDNIHLV 25878 MXRA5 247 SLDTLMTYV 22829 NLGN4Y 248 ALYGDIDAV 5743PTGS2 249 ALYGRLEVV 23294 ANKS1A 250 ALCEENMRGV 1938 EEF2 251SLLQATDFMSL 7070 THY1 252 YVYQNNIYL 2191 FAP 253 KLLDEVTYLEA 1573 CYP2J2254 VLFQEALWHV 2194 FASN 255 ALALWIPSL 200634 KRTCAP3 256 GLLEELVTV642475 MROH6 257 SLADFMQEV 23019 CNOT1 258 LLYEGKLTL 440107 PLEKHG7 259ALADKELLPSV 84883 AIFM2 260 ALLAEGITWV 54499 TMCO1 261 YLYDSETKNA 4316MMP7 262 VLAKPGVISV 1293 COL6A3 263 LLAGQTYHV 1293 COL6A3 264 RLLDVLAPLV80781 COL18A1 265 LLDKKIGV 10576 CCT2

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, lung cancer, brain cancer,stomach cancer, kidney cancer, liver cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma (MCC), melanoma,ovarian cancer, and esophageal cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 191. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 68 (see Table 1), and their uses in theimmunotherapy of CRC, lung cancer, brain cancer, stomach cancer, kidneycancer, liver cancer, pancreatic cancer, prostate cancer, leukemia,breast cancer, Merkel cell carcinoma, melanoma, ovarian cancer, andesophageal cancer, and preferably CRC.

Most preferred are the peptides—alone or in combination—selected fromthe group consisting of SEQ ID NO: 1, 3, 6, 11, 13, 16, 18, 19, 23, 24,26, 31, 32, 34, 37, 40, 44, 45, 59, 67, 71, 82, 87, 88, 100, 103, 105,113, 123, 124, 126, 129, 131, 132, 133, 135, 137, 140, 142, 150, 152,153, and SEQ ID NO: 166, and their uses in the immunotherapy of CRC,lung cancer, brain cancer, stomach cancer, kidney cancer, liver cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, and esophageal cancer, andpreferably CRC.

As shown in the following Table 4A and B, many of the peptides accordingto the present invention are also found on other tumor types and can,thus, also be used in the immunotherapy of other indications. Also referto FIG. 1D and Example 1.

TABLE 4A Peptides according to the present invention andtheir specific uses in other proliferative di-seases, especially in other cancerous diseases.The table shows for selected peptides on whichadditional tumor types they were found and eitherover-presented on more than 5% of the measuredtumor samples, or presented on more than 5% ofthe measured tumor samples with a ratio of geo-metric means tumor vs normal tissues being largerthan 3. Over-presentation is defined as higherpresentation on the tumor sample as compared tothe normal sample with highest presentation. SEQ IDOther relevant organs/ No. Sequence diseases   1 ALIKQLFEALung, Brain, Ovary, Esophagus   3 RLIPDTLYSVLung, Pancreas, Breast, Ovary,  Esophagus   4 RLAELTVDEFL Lung, Ovary  6 FLAELPGSLSL Lung, Liver, Leukocytes,  Melanoma, Ovary   8 ALMLQGVDLLPancreas, Leukocytes  10 RMYNKIFAI Liver  11 YLFEKTFNMLung, Brain, Esophagus  13 FLLAEDTKV Melanoma  15 LQLDKEFQLLung, Esophagus  16 VLVDQSWVL Ovary  18 FLSSLKGGLL Ovary  19 RLYTKLLNEABrain, Esophagus  25 SLADRLIGV Prostate, Ovary  26 GLASKENFSNVSLLung, Liver, Esophagus  29 QMLDVAIRV Leukocytes  31 LLYGKYVSVLung, Kidney, Brain, Liver,  Leukocytes, Ovary, Esophagus  33 ALWEKNTHLLiver, MCC  34 ILLEKSVSV Ovary  37 GLFAGLGGAGA Esophagus  38 SLAPTPVSAPancreas  40 ALSNVIHKV Lung, Pancreas, Esophagus  44 SLESKLTSVBrain, Pancreas, Ovary  45 ALAELLHGA Lung, Kidney, Brain, Liver, Prostate, Breast, Ovary  46 GLDDRYSLV Esophagus  47 KLYERCEVV Liver  48FLDASDPAL Kidney, Prostate  53 FLLGSEIKL Kidney, Pancreas  54ALLNGEYLLAA Liver, Ovary, Esophagus  55 QIITSVVSV Pancreas  56VLFTDEGVPKFL Lung, Kidney, Liver  57 NLLEKENYL Ovary  58 AMADKMDMSLBrain, Leukocytes, Melanoma  59 LLTDNVVKL Lung, Liver, Esophagus  61KLLKLFQGV Kidney  62 YLAPENGYL Lung, Liver, Melanoma,  Esophagus  63KLFSILSTV Brain, Liver, Prostate,  Ovary, Esophagus  65 FGAPGIISAStomach, Esophagus  67 SLNDLEKDVMLL Leukocytes, Melanoma  69GMLNEAEGKAIKL Prostate  70 MISELEVRL Kidney, Stomach, Prostate, Esophagus  71 RLVVTEIPTAI Liver  72 YLLDYPNNLLLung, Kidney, Brain, Liver,  Leukocytes, Breast, Ovary, Esophagus  74YLMGFLHAV Ovary  76 YLIGEKQHYL Liver  77 SLLKRDFGA Lung, Breast  79SLAADQLLKL Lung, Liver  80 QVDEVVDIMRV Leukocytes  81 ALLSQQTHLEsophagus  82 QLYEEPDTKL Leukocytes, Esophagus  83 LTIEDGIFEVKidney, Leukocytes, MCC,  Melanoma, Esophagus  84 SMVEDITGLRLLung, Liver, Esophagus  87 LLFDAPDLRL Lung, Ovary  88 KLDIKVETVLung, Kidney, Liver,  Melanoma, Ovary, Esophagus  89 SLIEYEFRVLiver, Esophagus  90 GLLKPGLNVVL Lung, Esophagus  91 TVDVATPSVBreast, Ovary  92 WIDDTSAFV Melanoma  98 AMNGKSFSV Liver, Esophagus 101GLSEGHTFQV Prostate 102 KILVSLIEV Lung, Kidney, Ovary,  Esophagus 103FLFGYPKRL Brain, Liver, Prostate 105 YALDLSTFL Kidney, Liver 107ALLGGGPYML Lung 108 SLAELVPGVGGI Kidney, Brain, Liver, Ovary 110LLGELPRLLLL Lung, Pancreas, Leukocytes 116 LVYQFVHPIPancreas, Prostate, Breast,  Ovary 117 TLQPVDNSTISLLung, Kidney, Liver, Pancreas,  Esophagus 118 LLADLKTMVBrain, Leukocytes, Melanoma 119 ILYQTVTGL Esophagus 121 SLAPNIISQLLiver, Leukocytes 123 KTLERSYLL Lung, Kidney, Liver, MCC, Ovary, Esophagus 124 RVLPPSALQSV Lung, Liver, MCC, Melanoma, Ovary, Esophagus 125 KLGDFGLLVEL Lung, Brain, Melanoma, Ovary, Esophagus 126 TLAKYLMEL Lung, Brain, Liver, Ovary,  Esophagus 127RLAELTVDEFLA Ovary 128 MLDDRAYLV Lung, Brain, Breast, MCC, Ovary, Esophagus 129 VLIDVLKEL Kidney, Leukocytes 131 KLLDVVHPALung, Brain, Liver, Prostate,  Ovary 132 ALLNAILHSALung, Brain, Liver, Ovary,  Esophagus 133 RTFEKIEEVLung, Kidney, Brain, Stomach,  Liver, Breast, MCC, Ovary, Esophagus 134GVAGGSILKGV Lung, Liver, Melanoma, Ovary,  Esophagus 135 KLQEEIPVL Lung136 KLFDIFSQQV Liver 137 QLTEIKPLL Brain, Ovary 138 KQFEGTVEI Esophagus139 VLLNEILEQV Lung, Liver, Melanoma, Ovary,  Esophagus 140 LLNEILEQVLung, Melanoma, Ovary 141 AVIEHLERL Lung, Kidney, Esophagus 142SLVQRVETI Lung, Kidney, Liver, Melanoma,  Ovary, Esophagus 143 KLSDVWKELLung 144 LLNDRIWLA Esophagus 145 LLLEVVKQV Melanoma, 146 ALSDETWGLKidney, Stomach, Pancreas,  Breast, Ovary 147 TLTELRAFL Kidney 148RLLENMTEVV Liver 149 YQFDKVGILTL Leukocytes, Melanoma 151 SAQGSDVSLTACKVLung 152 KLLAVIHEL Lung, Kidney, Pancreas,  Ovary, Esophagus 153ILFSEDSTKLFV Lung, Liver, Leukocytes,  Melanoma, Ovary, Esophagus 154KLPSETIFVGC Lung, Liver, Leukocytes,  Ovary, Esophagus 155 RLLGEEVVRVEsophagus 156 SLMMTIINL Lung, Liver, Melanoma 157 SLIERDLKLLung, Kidney, Brain, Liver,  Esophagus 158 GLLDPSVFHVKidney, Brain, Liver,  Esophagus 159 VLVDDDGIKVV Liver, Melanoma, Ovary160 KLLEFDQLQL Lung, Kidney, Leukocytes,  Ovary 161 FLKNELDNVLung, Liver, Leukocytes,  Breast, Melanoma, Ovary 162 KLMDYIDELBrain, Esophagus 163 RLLHEVQEL Brain 164 KMLDEILLQL Brain 165RLLDFPEAMVL Lung, Ovary 166 GLLEARGILGL Liver 167 SVIDHIHLISVLung, Melanoma, Ovary 168 GLIRFPLMTI Lung, Kidney, Liver 169 YLAHFIEGLBrain, Liver, Leukocytes,  Esophagus 170 ALAGGITMV Lung, Kidney, Liver, Pancreas, Melanoma,  Esophagus 171 RLQETEGMVAV Liver, Leukocytes, MCC172 LLLDTVTMQV Kidney, Melanoma, Ovary 173 KLGDLMVLL Leukocytes 174ILLDDNMQIRL Liver, Melanoma, Ovary 175 TLLGGKEAQALGV Ovary 177ALLQGAIESV Melanoma, Ovary, Esophagus 178 YLFREPATI Lung, Brain, Liver, Prostate, Melanoma, Ovary, Esophagus 180 NLLEIAPHLBrain, Leukocytes, Breast 181 NLFDLGGQYLRV Brain, Liver, Ovary 183TLQEVVTGV Prostate, Breast 184 SLLDENNVSSYL Lung, Kidney, Liver, Pancreas, Prostate, MCC, Melanoma, Ovary,  Esophagus 185 VLYTGVVRVLeukocytes, Melanoma,  Ovary 186 KMSEKILLL Esophagus 187 GLHNVVYGIProstate 188 FLVDGPRVQL Breast, Melanoma 189 AISEVIGKITA Ovary 190AMAEMVLQV Lung 191 QLFSEIHNL Brain, Liver 192 KIQEMQHFL Lung, Esophagus193 KLSPTVVGL Liver, Ovary 194 SLYKGLLSV Lung, Kidney, Brain, Liver, Ovary, Esophagus 195 LLLGERVAL Liver, Ovary 197 SLFGQDVKAVMCC, Esophagus 198 VLYGPDVPTI Pancreas 199 FLLEREQLLKidney, Leukocytes, Melanoma 200 SAVDFIRTL Breast, Esophagus 201GJFNGALAAV Brain, Pancreas 202 GLAALAVHL Melanoma, Ovary, Esophagus 203KLIDLSQVMYL Lung, Kidney, Pancreas, Ovary 204 KLLDLETERILLLung, Liver, Prostate, Ovary 205 RLHDENILL Lung, Kidney, Brain, Liver, Pancreas, Prostate, Ovary,  Esophagus 206 RIAGIRGIQGVLung, Kidney, Liver, Prostate,  Ovary 207 KLCEGFNEV Brain, Liver 208RLIDRIKTV Lung, Brain, Liver, Ovary 209 KLQDGLLHI Kidney, Brain, Liver, Pancreas 210 KLAVALLAA Lung, Kidney, Brain, Liver,  Esophagus 211SLFGKKYIL Kidney 212 FLLDGSANV Lung, Pancreas, Esophagus 214 SVLEKEIYSILung, Liver, Prostate,  Breast, Ovary, Esophagus 215 KLQEKIQELLung, Ovary, Esophagus 216 YLWDLDHGFAGV Lung, Brain, Liver, Prostate, Melanoma, Ovary, Esophagus 217 KLLDTMVDTFL Lung, Kidney, Brain, Liver, Ovary, Esophagus 218 KLSWDLIYL Lung, Kidney 220 KMDPVAYRVLiver, Prostate 221 ILNVDGLIGV Kidney, Brain, Liver, Prostate, Leukocytes 222 GVIAEILRGV Lung, Kidney, Brain, Liver 223VLMQDSRLYL Lung 224 QLQEGKNVIGL Pancreas 225 YLYGQTTTYLLung, Kidney, Stomach, Liver,  Melanoma, Ovary, Esophagus 226 FLVDGSWSVLung, Stomach, Pancreas,  Breast, Ovary, Esophagus 227 LTAPPEALLMVLung, Kidney, Brain, Liver,  Pancreas, Leukocytes, Ovary, Esophagus 228SMSGYDQVL Lung, Leukocytes 229 YLLEKFVAV Lung, Liver, Ovary 230AMSSKFFLV Lung, Brain, Stomach, Liver,  Pancreas, Prostate, Breast,Ovary, Esophagus 231 RLFADILNDV Lung, Brain, Liver, Prostate, MCC, Ovary 232 RLLDSVSRL Lung, Kidney, Liver, Pancreas, Breast, Ovary, Esophagus 233 RLDDLKMTV Lung, Kidney, Pancreas, Breast, Ovary, Esophagus 234 KMFESFIESV Lung, Kidney, Brain, Liver, Prostate, Ovary, Esophagus 235 LLHEENFSV Lung, Kidney, Liver, Ovary, Esophagus 236 KMSELQTYV Lung, Pancreas, Melanoma,  Ovary, Esophagus 237KLVEFDFLGA Lung, Brain, Stomach, Liver,  MCC, Ovary, Esophagus 238NMLEAVHTI Lung, Liver, Melanoma, Ovary,  Esophagus 239 QLIEKNWLLLung, Liver, Leukocytes,  Ovary, Esophagus 240 VLAPRVLRALung, Kidney, Brain, Liver,  Pancreas, Ovary 241 ILIDWLVQVLung, Kidney, Brain, Liver,  Pancreas, Ovary, Esophagus 242 RLEEDDGDVAMLung, Kidney, Brain, Liver,  Pancreas, Leukocytes, Breast, Melanoma 243TLMDMRLSQV Lung, Kidney, Brain, Liver,  Prostate, Ovary 244 SLHFLILYVLung, Kidney, Brain, Liver,  Melanoma 245 QLIDYERQLLung, Kidney, Liver,  Pancreas, Breast, Esophagus 246 GLTDNIHLVLung, Kidney, Pancreas,  Breast, Ovary, Esophagus 247 SLDTLMTYVLung, Kidney, Brain,  Pancreas, Prostate, Leukocytes, Esophagus 248ALYGDIDAV Lung, Brain, Pancreas,  Esophagus 249 ALYGRLEVVMCC, Ovary, Esophagus 250 ALCEENMRGV Lung, Kidney, Brain, Liver, MCC, Esophagus 251 SLLQATDFMSL Kidney, Pancreas, Esophagus 252 YVYQNNIYLLung, Stomach, Liver,  Pancreas, Breast, Melanoma, Ovary, Esophagus 253KLLDEVTYLEA Liver 254 VLFQEALWHV Liver 255 ALALWIPSLLung, Pancreas, Ovary,  Esophagus 256 GLLEELVTVLung, Stomach, Pancreas,  Ovary 257 SLADFMQEV Lung, Prostate, MCC, Ovary258 LLYEGKLTL Breast, Ovary 259 ALADKELLPSV Lung, Kidney, Liver, Pancreas, Prostate, Melanoma, Ovary, Esophagus 260 ALLAEGITVVV Liver 261YLYDSETKNA Kidney, Liver, Pancreas,  Ovary, Esophagus 262 VLAKPGVISVLung, Pancreas 264 RLLDVLAPLV Kidney, Liver 265 LLDKKIGVKidney, Ovary, Esophagus

TABLE 4B Peptides according to the present invention andtheir specific uses in other proliferative di-seases, especially in other cancerous diseases(amendment of Table 4A). The table shows, likeTable 4A, for selected peptides on which ad-ditional tumor types they were found showingover-presentation (including specific presenta-tion) on more than 5% of the measured tumorsamples, or presentation on more than 5% of themeasured tumor samples with a ratio of geometricmeans tumor vs normal tissues being larger than3. Over-presentation is defined as higher presen-tation on the tumor sample as compared to thenormal sample with highest presentation. Normaltissues against which over-presentation wastested were: adipose tissue, adrenal gland, bloodcells, blood vessel, bone marrow, brain,esophagus, eye, gallbladder, heart, kidney, largeintestine, liver, lung, lymph node, nerve, pan-creas, parathyroid gland, peritoneum, pituitary,pleura, salivary gland, skeletal muscle, skin,small intestine, spleen, stomach, thyroid gland,trachea, ureter, urinary bladder. SEQ ID NO. SequenceAdditional Entities   1 ALIKQLFEA SCLC, GC, BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL   2 ALLPRYFFL Uterine Cancer   3RLIPDTLYSV Melanoma, Urinary bladder  cancer, Uterine Cancer,Gallbladder Cancer, Bile  Duct Cancer   4 RLAELTVDEFLCLL, Melanoma, Urinary bladder  cancer, AML   6 FLAELPGSLSLSCLC, Urinary bladder cancer,  Uterine Cancer, GallbladderCancer, Bile Duct Cancer, AML,  NHL   8 ALMLQGVDLLBRCA, Melanoma, Urinary bladder  cancer, AML  11 YLFEKTFNMSCLC, Urinary bladder cancer  13 FLLAEDTKV Urinary bladder cancer, AML, NHL, OC  15 LQLDKEFQL CLL, BRCA, Urinary bladder cancer, Uterine Cancer, PC  16 VLVDQSVVVL Esophageal Cancer, Urinary bladder cancer  18 FLSSLKGGLL Melanoma, Urinary bladder cancer, Uterine Cancer, AML  19 RLYTKLLNEA Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer  22GLIDEVMVL Gallbladder Cancer, Bile  Duct Cancer  23 FLDANGHFVGC, Esophageal Cancer,  Gallbladder Cancer, Bile  Duct Cancer  25SLADRLIGV SCLC, BRCA, Uterine Cancer  26 GLASKENFSNVSUrinary bladder cancer,  L Uterine Cancer, GallbladderCancer, Bile Duct Cancer  28 ALTEIQEFI NSCLC, Brain Cancer, HCC, BRCA, Melanoma, Esophageal Cancer, Urinary bladder  cancer, GallbladderCancer, Bile Duct Cancer,  AML, NHL  29 QMLDVAIRV BRCA  31 LLYGKYVSVSCLC, Melanoma, Urinary  bladder cancer, Uterine  Cancer  32 KLNTETFGVGC, BRCA, Esophageal  Cancer, AML  33 ALWEKNTHL Urinary bladder cancer 34 ILLEKSVSV BRCA, Melanoma, Esophageal  Cancer, Urinary bladder cancer  35 KLLDLTVRI Gallbladder Cancer, Bile  Duct Cancer  36GLLESPSIFNFTA BRCA  37 GLFAGLGGAGA BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer,  Bile Duct Cancer 38 SLAPTPVSA Uterine Cancer  40 ALSNVIHKV Urinary bladder cancer  42SILDDSFKL Gallbladder Cancer, Bile  Duct Cancer  43 TLDAAQPRVPrC, Esophageal Cancer  44 SLESKLTSV Melanoma, Urinary bladder cancer, Uterine Cancer  45 ALAELLHGA Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer  46GLDDRYSLV Urinary bladder cancer  47 KLYERCEVV Melanoma  53 FLLGSEIKLHCC, Melanoma, Urinary  bladder cancer, AML  54 ALLNGEYLLAANSCLC, Brain Cancer, GC,  BRCA, Melanoma, Urinarybladder cancer, Uterine  Cancer, Gallbladder Cancer,  BileDuct Cancer, AML, NHL  55 QIITSVVSV CLL, Urinary bladder cancer, Uterine Cancer, AML  56 VLFTDEGVPKFL BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer, OC  58 AMADKMDMSL NSCLC, SCLC, BRCA, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL  59 LLTDNVVKL Melanoma, AML, NHL  61KLLKLFQGV Melanoma, AML  62 YLAPENGYL SCLC, CLL, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer, AML, NHL 63 KLFSILSTV SCLC, Melanoma, Urinary  bladder cancer, UterineCancer, Gallbladder Cancer,  Bile Duct Cancer, NHL  64 KTLGKLWRLMelanoma  66 GLDDGPDFL Melanoma  67 SLNDLEKDVMLL SCLC, Urinary bladder cancer, Uterine Cancer,  AML, NHL  71 RLVVTEIPTAINSCLC, SCLC, Melanoma,  Esophageal Cancer, Urinary bladder cancer  72YLLDYPNNLL SCLC, Melanoma, Urinary  bladder cancer, UterineCancer, Gallbladder Cancer,  Bile Duct Cancer, NHL, PC  74 YLMGFLHAVBRCA, Urinary bladder cancer  75 EMIENIQSV Gallbladder Cancer, Bile Duct Cancer  77 SLLKRDFGA SCLC, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, NHL  78 ALDPELLLL AML 79 SLAADQLLKL Uterine Cancer  80 QVDEVVDIMRV AML  81 ALLSQQTHLUrinary bladder cancer, AML  82 QLYEEPDTKL SCLC, Melanoma, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer,  AML, NHL  83LTIEDGIFEV NHL  84 SMVEDITGLRL SCLC, OC, Urinary bladder cancer, Uterine Cancer, NHL  87 LLFDAPDLRL SCLC, BRCA, Urinary bladder cancer, Uterine Cancer  88 KLDIKVETV BRCA, Urinary bladder cancer  90GLLKPGLNVVL Urinary bladder cancer, AML  91 TVDVATPSV CLL  93 SLQELRLLLSCLC  97 ALVWVGVVTV CLL  98 AMNGKSFSV NSCLC, SCLC, BRCA, Melanoma, OC, Urinary bladder cancer, Gallbladder Cancer, Bile  Duct Cancer  99KLLEVDLDTV Gallbladder Cancer, Bile  Duct Cancer, AML 100 SLDDFLATAPC, CLL, BRCA, Melanoma,  Esophageal Cancer, Urinarybladder cancer, Uterine  Cancer, Gallbladder Cancer,  Bile Duct Cancer102 KILVSLIEV NHL 103 FLFGYPKRL NSCLC, SCLC, Melanoma, Urinary bladder cancer, Uterine Cancer, AML 105 YALDLSTFLBRCA, Melanoma, Urinary  bladder cancer, PC 107 ALLGGGPYMLUrinary bladder cancer,  Gallbladder Cancer, Bile  Duct Cancer 108SLAELVPGVGGI BRCA 109 ALDGDQMEL AML 110 LLGELPRLLLLMelanoma, Esophageal Cancer,  Urinary bladder cancer 113 ILYDLQQNLSCLC, CLL, BRCA, Melanoma,  Urinary bladder cancer,Uterine Cancer, Gallbladder  Cancer, Bile Duct Cancer, AML, NHL 114TAVGHALVL BRCA, Melanoma, Gallbladder  Cancer, Bile Duct Cancer 116LVYQFVHPI Urinary bladder cancer,  Uterine Cancer, GallbladderCancer, Bile Duct Cancer 117 TLQPVDNSTISL Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 118 LLADLKTMV NHL 119 ILYQTVTGLCLL, Melanoma, Urinary bladder  cancer, Uterine Cancer, AML, NHL 120VLYEGVDEV SCLC, BRCA, Gallbladder Cancer,  Bile Duct Cancer 121SLAPNIISQL AML 123 KTLERSYLL SCLC, BRCA, Urinary bladder cancer, Uterine Cancer, AML, NHL, PC 124 RVLPPSALQSVSCLC, BRCA, Urinary bladder  cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct  Cancer, AML, NHL, PC 125 KLGDFGLLVELSCLC, Urinary bladder cancer,  AML, PC 126 TLAKYLMELSCLC, BRCA, Melanoma, Urinary  bladder cancer, UterineCancer, Gallbladder Cancer,  Bile Duct Cancer, AML, NHL 127 RLAELTVDEFLASCLC, Melanoma, Urinary bladder  cancer, Uterine Cancer, AML 128MLDDRAYLV PC 129 VLIDVLKEL Melanoma, NHL 130 GLGGSQLIDTHLEsophageal Cancer, Uterine  Cancer 131 KLLDVVHPACLL, BRCA, Urinary bladder  cancer, Uterine Cancer, AML,  NHL 132ALLNAILHSA SCLC, CLL, Melanoma, Urinary  bladder cancer, UterineCancer, NHL, PC 133 RTFEKIEEV SCLC, Melanoma, Urinary bladder cancer, Uterine Cancer, AML, NHL 134 GVAGGSILKGVCLL, BRCA, Urinary bladder  cancer, Gallbladder Cancer,Bile Duct Cancer, NHL 135 KLQEEIPVL BRCA, Melanoma, NHL 136 KLFDIFSQQVUrinary bladder cancer,  Uterine Cancer, NHL 137 QLTEIKPLLCLL, Urinary bladder cancer,  Uterine Cancer, GallbladderCancer, Bile Duct Cancer,  AML, NHL 138 KQFEGTVEI CLL, NHL 139VLLNEILEQV SCLC, CLL, Urinary bladder  cancer, Uterine Cancer, AML,NHL, PC 140 LLNEILEQV SCLC, CLL, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer, AML, NHL142 SLVQRVETI SCLC, PC, BRCA, Urinary  bladder cancer, UterineCancer, Gallbladder Cancer,  Bile Duct Cancer, NHL 143 KLSDVWKELGallbladder Cancer, Bile  Duct Cancer 144 LLNDRIWLABRCA, Melanoma, Uterine  Cancer 145 LLLEVVKQV Gallbladder Cancer, Bile Duct Cancer, NHL 146 ALSDETWGL SCLC, CLL, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 147 TLTELRAFL CLL, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer, NHL 148 RLLENMTEVVCLL, OC, Urinary bladder  cancer, Uterine Cancer, NHL 149 YQFDKVGILTLSCLC, RCC, Brain Cancer 150 RLADLEALKV Urinary bladder cancer, NHL 152KLLAVIHEL BRCA, Melanoma, Urinary  bladder cancer, GallbladderCancer, Bile Duct Cancer,  AML, NHL 153 ILFSEDSTKLFVUrinary bladder cancer, NHL 154 KLPSETIFVGCMelanoma, Uterine Cancer, AML 155 RLLGEEVVRV Melanoma 156 SLMMTIINLSCLC, GC, Urinary bladder  cancer, Uterine Cancer,Gallbladder Cancer, Bile  Duct Cancer, AML, NHL, OC 158 GLLDPSVFHVMelanoma, Urinary bladder  cancer, Gallbladder Cancer,Bile Duct Cancer, AML, NHL 159 VLVDDDGIKVVSCLC, BRCA, Esophageal Cancer,  Urinary bladder cancer,Uterine Cancer, Gallbladder  Cancer, Bile Duct Cancer, NHL 160KLLEFDQLQL SCLC 161 FLKNELDNV Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, AML, NHL 162 KLMDYIDELNSCLC, BRCA, Melanoma,  Urinary bladder cancer,Gallbladder Cancer, Bile Duct  Cancer, NHL 163 RLLHEVQEL RCC, AML, NHL164 KMLDEILLQL SCLC, RCC, CLL, Melanoma, OC,  Urinary bladder cancer,AML, NHL 165 RLLDFPEAMVL SCLC, CLL, Urinary bladder cancer, Uterine Cancer 166 GLLEARGILGL Urinary bladder cancer, AML,  NHL167 SVIDHIHLISV SCLC, BRCA 168 GLIRFPLMTICLL, Melanoma, Urinary bladder  cancer, Uterine Cancer, AML 169YLAHFIEGL Urinary bladder cancer, OC 170 ALAGGITMV CLL, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 171 RLQETEGMVAVMelanoma, OC 172 LLLDTVTMQV Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, AML 173 KLGDLMVLLMelanoma, AML, NHL 174 ILLDDNMQIRL SCLC, CLL, Urinary bladder cancer, AML 177 ALLQGAIESV SCLC, GC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 178 YLFREPATIBRCA, Urinary bladder cancer,  Uterine Cancer, PC 179 RLLJPLSSAAML, BRCA, PC, Gallbaldder  Cancer, HCC, Melanoma,NHL, OC, Esophageal Cancer,  Brain Cancer, NSCLC, SCLC, Uterine Cancer180 NLLEIAPHL NSCLC, Melanoma, OC,  Esophageal Cancer, Urinarybladder cancer, Uterine  Cancer, Gallbladder Cancer, Bile Duct Cancer, AML 181 NLFDLGGQYLRV CLL, Melanoma, Urinary bladder cancer 182 SLNKWIFTV Melanoma 183 TLQEVVTGVCLL, Melanoma, Urinary  bladder cancer, Uterine  Cancer, NHL 184SLLDENNVSSYL SCLC, CLL, BRCA, Urinary  bladder cancer, GallbladderCancer, Bile Duct Cancer,  AML, NHL, OC 185 VLYTGVVRVSCLC, BRCA, Esophageal  Cancer, AML, NHL 186 KMSEKILLL Melanoma 187GLHNVVYGI CLL, Melanoma, Urinary  bladder cancer, NHL 188 FLVDGPRVQLCLL, Uterine Cancer 189 AISEVIGKITA Gallbladder Cancer, Bile Duct Cancer, PC 190 AMAEMVLQV SCLC, CLL, BRCA, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer,  AML, NHL 191QLFSEIHNL SCLC, Melanoma, Urinary  bladder cancer, GallbladderCancer, Bile Duct Cancer,  AML, NHL, PC NSCLC = non-small cell lungcancer, SCLC = small cell lung cancer, RCC = kidney cancer, CRC = colonor rectum cancer, GC = stomach cancer, HCC = liver cancer, PC =pancreatic cancer, PrC = prostate cancer, BRCA = breast cancer, MCC =Merkel cell carcinoma, OC = ovarian cancer, NHL = non-Hodgkin lymphoma,AML = acute myeloid leukemia, CLL = chronic lymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 3, 4, 6, 11, 15, 26, 31, 40, 45, 56, 59, 62, 72,77, 79, 84, 87, 88, 90, 102, 107, 110, 117, 123, 124, 125, 126, 128,131, 132, 133, 134, 135, 139, 140, 141, 142, 143, 151, 152, 153, 154,156, 157, 160, 161, 165, 167, 168, 170, 178, 184, 190, 192, 194, 203,204, 205, 206, 208, 210, 212, 214, 215, 216, 217, 218, 222, 223, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 252, 255, 256, 257,259, and 262 for the—in one preferred embodiment combined—treatment oflung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 11, 19, 31, 44, 45, 58, 63, 72, 103, 108, 118, 125,126, 128, 131, 132, 133, 137, 157, 158, 162, 163, 164, 169, 178, 180,181, 191, 194, 201, 205, 207, 208, 209, 210, 216, 217, 221, 222, 227,230, 231, 234, 237, 240, 241, 242, 243, 244, 247, 248, and 250 forthe—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 65, 70, 133, 146, 225, 226, 230, 237, 252, and 256 forthe—in one preferred embodiment combined—treatment of stomach cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 31, 45, 48, 53, 56, 61, 70, 72, 83, 88, 102, 105, 108,117, 123, 129, 133, 141, 142, 146, 147, 152, 157, 158, 160, 168, 170,172, 184, 194, 199, 203, 205, 206, 209, 210, 211, 217, 218, 221, 222,225, 227, 232, 233, 234, 235, 240, 241, 242, 243, 244, 245, 246, 247,250, 251, 259, 261, 264, and 265 for the—in one preferred embodimentcombined—treatment of kidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6, 10, 26, 31, 33, 45, 47, 54, 56, 59, 62, 63, 71, 72,76, 79, 84, 88, 89, 98, 103, 105, 108, 117, 121, 123, 124, 126, 131,132, 133, 134, 136, 139, 142, 148, 153, 154, 156, 157, 158, 159, 161,166, 168, 169, 170, 171, 174, 178, 181, 184, 191, 193, 194, 195, 204,205, 206, 207, 208, 209, 210, 214, 216, 217, 220, 221, 222, 225, 227,229, 230, 231, 232, 234, 235, 237, 238, 239, 240, 241, 242, 243, 244,245, 250, 252, 253, 254, 259, 260, 261, and 264 for the—in one preferredembodiment combined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 3, 8, 38, 40, 44, 53, 55, 110, 116, 117, 146, 152,170, 184, 198, 201, 203, 205, 209, 212, 224, 226, 227, 230, 232, 233,236, 240, 241, 242, 245, 246, 247, 248, 251, 252, 255, 256, 259, 261,and 262 for the—in one preferred embodiment combined—treatment ofpancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 25, 45, 48, 63, 69, 70, 101, 103, 116, 131, 178, 183,184, 187, 204, 205, 206, 214, 216, 220, 221, 230, 231, 234, 243, 247,257, and 259 for the—in one preferred embodiment combined—treatment ofprostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6, 8, 29, 31, 58, 67, 72, 80, 82, 83, 110, 118, 121,129, 149, 153, 154, 160, 161, 169, 171, 173, 180, 185, 199, 221, 227,228, 239, 242, and 247 for the—in one preferred embodimentcombined—treatment of leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 3, 45, 72, 77, 91, 116, 128, 133, 146, 161, 180, 183,188, 200, 214, 226, 230, 232, 233, 242, 245, 246, 252, and 258 forthe—in one preferred embodiment combined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 33, 83, 123, 124, 128, 133, 171, 184, 197, 231, 237,249, 250, and 257 for the—in one preferred embodiment combined—treatmentof Merkel cell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6, 13, 58, 62, 67, 83, 88, 92, 118, 124, 125, 134,139, 140, 142, 145, 149, 153, 156, 159, 161, 167, 170, 172, 174, 177,178, 184, 185, 188, 199, 202, 216, 225, 236, 238, 242, 244, 252, and 259for the—in one preferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 3, 4, 6, 16, 18, 25, 31, 34, 44, 45, 54, 57, 63,72, 74, 87, 88, 91, 102, 108, 116, 123, 124, 125, 126, 127, 128, 131,132, 133, 134, 137, 139, 140, 142, 146, 152, 153, 154, 159, 160, 161,165, 167, 172, 174, 175, 177, 178, 181, 184, 185, 189, 193, 194, 195,202, 203, 204, 205, 206, 208, 214, 215, 216, 217, 225, 226, 227, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 246,249, 252, 255, 256, 257, 258, 259, 261, and 265 for the—in one preferredembodiment combined—treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 3, 11, 15, 19, 26, 31, 37, 40, 46, 54, 59, 62, 63,65, 70, 72, 81, 82, 83, 84, 88, 89, 90, 98, 102, 117, 119, 123, 124,125, 126, 128, 132, 133, 134, 138, 139, 141, 142, 144, 152, 153, 154,155, 157, 158, 162, 169, 170, 177, 178, 184, 186, 192, 194, 197, 200,202, 205, 210, 212, 214, 215, 216, 217, 225, 226, 227, 230, 232, 233,234, 235, 236, 237, 238, 239, 241, 245, 246, 247, 248, 249, 250, 251,252, 255, 259, 261, and 265 for the—in one preferred embodimentcombined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofCRC, lung cancer, brain cancer, stomach cancer, kidney cancer, livercancer, pancreatic cancer, prostate cancer, leukemia, breast cancer,Merkel cell carcinoma, melanoma, ovarian cancer, and esophageal cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 191.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the

HLA-DR antigen-associated invariant chain 00, or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T-cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 191, preferably containing SEQ IDNo. 1 to SEQ ID No. 68, or a variant amino acid sequence.

The present invention further relates to activated T-cells, produced bythe method according to the present invention, wherein said T-cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T-cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T-cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are CRC, lung cancer, brain cancer,stomach cancer, kidney cancer, liver cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma (MCC), melanoma,ovarian cancer, and esophageal cancer, and preferably CRC cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably CRC. The markercan be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

AKAP8 (also called AKAP95) encodes a member of the A-kinase anchorprotein family, which includes scaffold proteins that contain a bindingdomain for the RI/RII subunit of protein kinase A (PKA) and recruit PKAand other signaling molecules to specific subcellular locations. AKAP8binds to the RII alpha subunit of PKA and may play a role in chromosomecondensation during mitosis by targeting PKA and the condensin complexto chromatin (RefSeq, 2002). AKAP8 protein expression is significantlyup-regulated in rectal and lung cancer and is associated with celldifferentiation and the histopathological type, suggesting an importantrole in tumor development and progression. The expression of AKAP8correlates with the expression of Cyclin E and Cyclin D (Chen et al.,2012; Hu et al., 2013; Qi et al., 2015).

ARHGAP39 (also called Vilse or CrGAP) encodes a Rho GTPase-activatingprotein that is involved in Roundabout (Robo) receptor-mediatedrepulsive axon guidance and the regulation of RAC-dependent cytoskeletalchanges (Hu et al., 2005). ARHGAP39 is frequently up-regulated inbladder cancer cell lines (Matsuda et al., 2011). ARHGAP39 is involvedin directional migration of endothelial cells (Kaur et al., 2008).

AURKB (also called AIM-1) encodes aurora kinase B a member of the aurorakinase subfamily of serine/threonine kinases. AURKB regulates togetherwith other proteins the segregation of chromosomes during mitosis andmeiosis through association with microtubules (RefSeq, 2002). AURKBexpression is up-regulated in different cancer types, including lung,colorectal and breast cancer as well as leukemia and thereby associatedwith poor prognosis. So development of AURKB inhibitors for clinicaltherapy is an interesting field (Hayama et al., 2007; Pohl et al., 2011;Hegyi et al., 2012; Goldenson and Crispino, 2015). AURKB over-expressionleads to phosphorylation of histone H3 and to chromosome instability, acrucial factor for carcinogenesis (Ota et al., 2002; Tatsuka et al.,1998). AURKB activity augments the oncogenic Ras-mediated celltransformation (Kanda et al., 2005).

C18orf21 (also called XTP13) encodes an abnormal hemoglobin beta chainpeptide associated with inhibition of HIV infection (Liu et al., 2011a;Aschauer et al., 1983).

CCNB1 encodes for cyclin B1 a regulatory protein involved in mitosis. Ithas two alternative transcripts, one expressed constitutively and theother predominantly during G2/M phase (RefSeq, 2002). CCNB1 encodescyclin B1, a regulatory protein involved in mitosis (RefSeq, 2002).CCNB1 is a well-described tumor antigen and CCNB1 over-expression hasbeen described for breast, head and neck, prostate, colorectal, lung andliver cancers (Egloff et al., 2006). CCNB1 was shown to be up-regulatedin a variety of cancer entities, including colorectal cancer, breastcancer, lung cancer and renal cancer. The down-regulation of CCNB1 leadsto G2/M phase cell cycle arrest and the inhibition of proliferation andmigration (Chang et al., 2013; Sakurai et al., 2014; Fang et al., 2014;Ding et al., 2014). Genetic polymorphisms in the CCNB1 gene are relatedwith breast cancer susceptibility, progression and survival of ChineseHan woman (Li et al., 2013).

CCNB2 encodes cyclin B2, a member of the family of cyclins that plays arole in cell cycle regulation (RefSeq, 2002). CCNB2 is up-regulated incolorectal adenocarcinoma (Park et al., 2007). CCNB2 is over-expressedin various human tumors. Strong CCNB2 expression in tumor cells isassociated with a poor prognosis in patients with adenocarcinoma of lungand invasive breast carcinoma (Takashima et al., 2014; Albulescu, 2013).

CCT7 encodes for chaperonin containing TCP1 complex (CCT) subunit 7(eta), which is involved in the ATP-dependent folding of variousproteins including actin and tubulin. CCT7 was found to be a part of aprotein sub-network, which is significantly discriminative of late stagehuman colorectal cancer (Nibbe et al., 2009).

CDC42BPG (also called MRCKgamma) encodes CDC42 Binding Protein KinaseGamma (DMPK-Like), a member of the myotonic dystrophy kinase-relatedCDC42 binding kinases family (Ng et al., 2004).

CDC6 encodes a protein essential for the initiation of DNA replication(RefSeq, 2002). CDC6 expression is de-regulated in different cancertypes including gallbladder, cervical and prostate cancer (Wu et al.,2009; Wang et al., 2009c; Robles et al., 2002; Shu et al., 2012). CDC6co-operates with c-Myc to promote genetic instability, tumor-liketransformation and apoptosis attenuation (Chen et al., 2014a).Hypoxia-induced ATR promotes the degradation of CDC6. Initiation of DNAreplication is regulated by p53 through Cdc6 protein stability (Duursmaand Agami, 2005; Martin et al., 2012).

CENPE encodes centromere protein E, 312 kDa, a kinesin-like motorprotein that accumulates in the G2 phase of the cell cycle. CENPE isproposed to be one of the motors responsible for mammalian chromosomemovement and/or spindle elongation (RefSeq, 2002). CENPE expressionsignificantly correlated with glioma grade and might complement otherparameters for predicting survival time for glioma patients (Bie et al.,2011). CENPE is up-regulated in chemo-resistant epithelial ovariantumors compared to chemo-sensitive tumors (Ju et al., 2009). CENPE isup-regulated in invasive and aggressive-invasive prolactin pituitarytumors (Werinckx et al., 2007).

CIRH1A (also called Cirhin) encodes cirrhosis autosomal recessive 1 A, aWD40-repeat-containing protein localized in the nucleolus. It causesNorth American Indian childhood cirrhosis (NAIC) (RefSeq, 2002). CIRH1Acan up-regulate a canonical NF-kappaB element and might participate inthe regulation of other genes containing NF-kappaB elements. Thissuggests that CIRH1A can influence the cancer-related NF-kappaB pathway(Yu et al., 2009).

CNOT1 encodes an enzymatic relevant subunit of the CCR4-NOT deadenylasecomplex which is an important regulator of translation and mRNAstability (Ito et al., 2011; Boland et al., 2013).

Single-nucleotide polymorphisms (SNPs) in the CNOT1 gene were detectedin osteosarcoma and acute lymphoblastic leukemia (ALL) (Gutierrez-Caminoet al., 2014; Bilbao-Aldaiturriaga et al., 2015). CNOT1 depletioninduces stabilization of mRNAs and activation of ER stress-mediatedapoptosis (Ito et al., 2011).

COL12A1 encodes the alpha chain of type XII collagen, a member of theFACIT (fibril-associated collagens with interrupted triple helices)collagen family and thus is a part of extracellular matrix (ECM)(RefSeq, 2002). COL12A1 is over-expressed in drug-resistant variants ofovarian cancer cell lines (Januchowski et al., 2014). In colorectalcancer, COL12A1 is over-expressed in desmoplastic stroma by and aroundcancer-associated fibroblasts, as well as in cancer cells lining theinvasion front (Karagiannis et al., 2012).

CYP2W1 encodes a member of the cytochrome P450 superfamily of enzymeswhich are monooxygenases catalyzing many reactions involved in drugmetabolism and in the synthesis of cholesterol, steroids and otherlipids (RefSeq, 2002). CYP2W1 is over-expressed in a variety of humancancers including hepatocellular, colorectal and gastric cancer. CYP2W1over-expression is associated with tumor progression and poor survival(Aung et al., 2006; Gomez et al., 2010; Zhang et al., 2014e). Due totumor-specific expression, CYP2W1 is an interesting drug target orenzymatic activator of pro-drugs during cancer therapy (Karlgren andIngelman-Sundberg, 2007; Nishida et al., 2010).

ECT2 encodes the epithelial cell transforming protein 2, a guaninenucleotide exchange factor and transforming protein that is related toRho-specific exchange factors and cell cycle regulators (RefSeq, 2002).ECT2 is over-expressed as a result of tumor-specific gene amplificationsin a variety of human tumors including lung, ovarian, gastric andpancreatic cancer. ECT2 is important for cell proliferation, migration,invasion and tumorigenicity (Fields and Justilien, 2010; Jin et al.,2014). Protein kinase C iota and ECT2 activate through MEK/ERK signalinga tumor-initiating cell phenotype in ovarian cancer (Wang et al.,2013e). Nuclear ECT2 is binding preferentially to the Rho GTPase Rac1and leads through Rac1 activation to cellular transformation, whilecytoplasmic ECT2 binds to the Rho GTPase RhoA and leads through RhoAactivation to the formation of cytokinetic furrow (Su et al., 2011; Huffet al., 2013).

GPR56 encodes adhesion G protein-coupled receptor G1 (ADGRG1), whichregulates brain cortical patterning. GPR56 binds specifically totransglutaminase 2, an inhibitor of tumor progression (RefSeq, 2002).GPR56 inhibits tumorigenesis by suppression of tumor growth andmetastasis in melanomas and prostate cancer. The role in other cancertypes appeared to be complex, maybe due to the varying ability of thedifferent splicing variants of GPR56 to activate transcription factorslike for c-myc and p53 response elements (Kim et al., 2010b; Xu et al.,2010; Yang and Xu, 2012). GPR56 inhibits VEGF production from melanomacells and impedes their angiogenesis and growth through a signalingpathway involving protein kinase C alpha (Yang et al., 2011a).

HS6ST2 encodes a member of the heparin sulfate (HS) sulfotransferasegene family. HS6ST2 catalyzes the transfer of sulfate to HS (RefSeq,2002). HS6ST2 is over-expressed in different cancer types includingthyroid, colorectal and ovarian cancer and is associated with migration,invasion and poor prognosis (Backen et al., 2007; Hatabe et al., 2013;Di et al., 2014). TGF-beta promotes cancer metastasis by stimulation ofHS6ST2 and IL-11 production (Pollari et al., 2012). HS6ST2 is aregulator of angiogenesis in response to EGF, FGF2 and VEGF signalingpathways (Ferreras et al., 2012; Cole et al., 2014).

IER3 (also called IEX-1) encodes immediate early response 3 that has afunction in protection of cells from Fas- or tumor necrosis factoralpha-induced apoptosis (RefSeq, 2002). De-regulation of IER3 expressionin ovarian, pancreatic, blood, breast and colorectal cancer, lymphomaand myeloma is linked to poor or better prognosis, depending on the typeand progression stages of tumors and makes the protein a valuablebiomarker, either alone or with other genes (Wu et al., 2013). IER3 geneexpression plays an important role in regulating apoptosis and cellgrowth through a positive or negative way. Over-expression of IER3renders some cells sensitive to apoptosis and accelerates cell cycleprogression, but reduces proliferation of other cells, whereasdisruption of IER3 expression is associated with a decrease in bothapoptosis and cell cycle progression (Zhang et al., 2011a). IER3interferes with certain signaling pathways, in particular NF-kappaB,MAPK/ERK and P13K/Akt. Mouse models also revealed an involvement of IER3expression in immune functions (Arlt and Schafer, 2011; Wu, 2003).

ITPR3 encodes a receptor for inositol 1,4,5-trisphosphate containing aC-terminal calcium-channel and a N-terminal ligand-binding site. ITPR3plays a role in exocrine secretion underlying energy metabolism andgrowth (RefSeq, 2002). ITPR3 is over-expressed in several cancer typesincluding colorectal, gastric and breast cancer and directly related tocancer progression and the aggressiveness of the tumor (Shibao et al.,2010; Mound et al., 2013; Sakakura et al., 2003). Akt can protect cellsin an ITPR3-dependent manner from apoptosis through reducing the Ca2+release from the endoplasmatic reticulum (Marchi et al., 2012).

KCNN4 (also called KCa3.1 or hIKCa1) encodes a part of aheterotetrameric voltage-independent potassium channel that is activatedby intracellular calcium. The activation of this channel is followed bymembrane hyper-polarization which promotes calcium influx (RefSeq,2002). KCNN4 is up-regulated in several cancers including breast, lungand prostate cancer and is associated with cell proliferation and tumorgrowth (Chou et al., 2008; Lallet-Daher et al., 2009; Haren et al.,2010; Bulk et al., 2015). Inhibition of KCNN4 regulates reactive oxygenspecies (ROS) levels and promotes p53 activation which suppresses thegrowth and migration of cells and leads to apoptosis (Liu et al.,2015b).

KIRREL (also called NEPH1) encodes a member of the nephrin-like proteinfamily whose members interact with the cytoplasmic domain of podocin(RefSeq, 2002).

KLK10 (also called NES1) encodes a member of the kallikrein subfamily ofserine proteases which play a role in carcinogenesis and have potentialas biomarkers (RefSeq, 2002). KLK10 is up-regulated in colon, ovarianand gastric cancer but down-regulated in breast, lung and prostatecancer (Yousef et al., 2005; Feng et al., 2006; Zhang et al., 2010; Liet al., 2001). The epigenetic silencing of KLK10 is maintained byTGFbeta/Smad signaling whereas KLK10 up-regulation is promoted byactivate Ras/MEK/ERK and PI3K/Akt signaling (Paliouras and Diamandis,2008; Papageorgis et al., 2010).

LIG1 is a DNA repair gene involved in the nucleotide excision repair(NER) and the base excision repair (BER) pathways. LIG1single-nucleotide polymorphisms are associated with the risk of lungcancer, endometrial cancer and glioma (Doherty et al., 2011; Lee et al.,2008b; Liu et al., 2009).

LSG1 encodes large 60S subunit nuclear xxport GTPase 1. The protein isnecessary for cell viability and may localize in the endoplasmicreticulum, nucleus and cytoplasm (RefSeq, 2002).

LSM14B (also called RAP55B) encodes a member of the LSM (like Sm) domainfamily that is involved in RNA metabolism, regulation of the mitoticG2/M phase, translational repression, incorporation into mRNP particles,P-body formation and stress granule localization (Marnef et al., 2009;Albrecht and Lengauer, 2004).

MAGED2 encodes melanoma antigen family D, 2, a member of a new definedMAGE-D cluster in Xp11.2, a hot spot for X-linked mental retardation.MAGED2 is expressed ubiquitously with high expression levels in specificbrain regions and in the interstitium of testes. MAGED2 is a potentialnegative regulator of wildtype p53 activity (Langnaese et al., 2001;Papageorgio et al., 2007). MAGED2 over-expression is associated withmelanoma, breast cancer and colon cancer (Li et al., 2004; Strekalova etal., 2015).

MAGEF1 encodes a member of the melanoma antigen (MAGE) superfamily thatcontains a microsatellite repeat and is ubiquitously expressed,suggesting a role in normal cell physiology (Stone et al., 2001).Flavopiridol induces an inhibition of human tumor cell proliferation andthe down-regulation of MAGEF1 in different human tumor cell lines (Lu etal., 2004). MAGEF1 is significantly over-expressed in colorectal cancertissues (Chung et al., 2010).

MDH1 encodes malate dehydrogenase, an enzyme that catalyzes thereversible oxidation of malate to oxaloacetate utilizing the NAD/NADHcofactor system in the citric acid cycle. MDH1 is localized to thecytoplasm and may play a pivotal role in the malate-aspartate shuttlewhich operates in the metabolic coordination between cytosol andmitochondria (RefSeq, 2002). In glioblastoma MDH1 is a target forseveral de-regulated microRNAs and its expression is repressed. Togetherwith known tumor suppressors or oncogenes MDH1 can help to discriminatelow versus high grade gliomas (Lages et al., 2011; Kounelakis et al.,2013). MDH1 is over-expressed in null cell pituitary adenomas and inthyroid oncocytomas (Hu et al., 2007; Baris et al., 2004).

MYO10 encodes a member of the myosin superfamily that represents anunconventional myosin. It functions as an actin-based molecular motorand plays a role in the integration of F-actin and microtubulecytoskeletons during meiosis (RefSeq, 2002). MYO10 is over-expressed inseveral cancer entities, including breast and lung cancer and isassociated with metastasis, cell migration and an aggressive phenotype(Cao et al., 2014; Sun et al., 2015b; Courson and Cheney, 2015). Mutantp53 promotes

NCAPG encodes the non-SMC condensing I complex subunit G which isresponsible for the condensation and stabilization of chromosomes duringmitosis and meiosis (RefSeq, 2002). NCAPG is down-regulated in patientswith multiple myeloma, acute myeloid leukemia, and leukemic cells fromblood or myeloma cells (Cohen et al., 2014). NCAPG may be a multi-drugresistant gene in colorectal cancer (Li et al., 2012). NCAPG is highlyup-regulated in the chromophobe subtype of human cell carcinoma but notin conventional human renal cell carcinoma (Kim et al., 2010a).Up-regulation of NCAPG is associated with melanoma progression (Ryu etal., 2007). NCAPG is associated with uveal melanoma (Van Ginkel et al.,1998). NCAPG shows variable expression in different tumor cells (Jageret al., 2000).

NDRG3 encodes a member of N-myc downstream-regulated genes that ishighly expressed in testis, prostate and ovary and may play a role inspermatogenesis (Zhao et al., 2001). NDRG3 may function as a tumorsuppressor gene in different cancer types including bladder cancer (Yanget al., 2013; Tsui et al., 2015). NDRG3 acts as a tumor promoter inprostate cancer where an up-regulated expression leads to an increasedgrowth rate, higher migration and induction of angiogenic chemokines.Up-regulation of NDRG3 is associated with a malignant phenotype inhepatocellular cancer cells (Wang et al., 2009b; Fan et al., 2011).

NOL11 encodes nucleolar protein 11 that is required for optimal rDNAtranscription in the ribosome biogenesis (Freed et al., 2012; Griffin etal., 2015). No111 is an interactor of the breast and ovarian tumorsuppressor BRCA1 (Hill et al., 2014).

PLAGL2 encodes a member of pleiomorphic adenoma gene (PLAG) family andis a zinc finger protein that recognizes DNA and/or RNA (Kas et al.,1998). PLAGL2 functions as a proto-oncogene in a variety of cancersincluding leukemia, gliomas, colorectal cancer and lung adenocarcinomas.There is also evidence that PLAGL2 can act as a tumor suppressor byinitiating cell cycle arrest and apoptosis (Yang et al., 2011b; Hanksand Gauss, 2012; Liu et al., 2014a). PLAGL2 prevents proteosomaldegradation of the E3 ubiquitin ligase Pirh2 which is regulating thestability of p53. PLAGL2 expression also increases the p73 level andup-regulates p73 target genes like p21 and Bax (Zheng et al., 2007;Hanks and Gauss, 2012; Landrette et al., 2005).

PTCD2 encodes the pentatricopeptide repeat domain protein 2 that may beinvolved in processing RNA transcripts, including cytochrome b derivedfrom mitochondrial DNA. Dysfunction of this protein plays a possiblerole in the etiology of heart failure (Xu et al., 2008).

RAD54 encodes a protein belonging to the DEAD-like helicase superfamily.It shares similarity with Saccharomyces cerevisiae RAD54 and RDH54, bothof which are involved in homologous recombination and repair of DNA.This protein binds to double-stranded DNA, and displays ATPase activityin the presence of DNA. This gene is highly expressed in testis andspleen, which suggests active roles in meiotic and mitotic recombination(RefSeq, 2002). Homozygous mutations of RAD54B were observed in primarylymphoma and colon cancer (Hiramoto et al., 1999). RAD54B counteractsgenome-destabilizing effects of direct binding of RAD51 to dsDNA inhuman tumor cells (Mason et al., 2015).

RNASEH2A encodes a component of the heterotrimeric type II ribonucleaseH enzyme and is the major source of its activity. RNASEH2A is anendonuclease and is predicted to remove Okazaki fragment RNA primersduring lagging strand DNA synthesis (RefSeq, 2002). RNASEH2A isup-regulated in transformed mesenchymal stem cells and over-expressed innumerous cancer cells, including aggressive prostate cancer. Knock-downof RNASEH2A inhibits anchorage-independent growth but does not alterproliferation of cancer cells (Flanagan et al., 2009; Williams et al.,2014).

RRM1 encodes ribonucleotide reductase M1 an enzyme that is essential forthe production of deoxyribonucleotides prior to DNA synthesis in S phaseof dividing cells. It is one of several genes located in the imprintinggene domain of 11p15.5, an important tumor suppressor gene region(RefSeq, 2002). RRM1 is involved in the regulation of cellproliferation, cell migration, tumorigenesis and metastasis development.Studies with large numbers of patients with different types of cancer,such as lung, pancreatic, breast and gastric cancer establish theprognostic or predictive value of RRM1 (Carvalho et al., 2009; Jordheimet al., 2011; Wang et al., 2013d). The nucleoside analog gemcitabine, acommon chemotherapeutic in cancer treatment, is targeting RRM1 (Jordheimand Dumontet, 2013).

SERPINB5 (also called maspin) encodes serpin peptidase inhibitor clade Bmember 5 that is characterized as a class II tumor suppressor based onits ability to promote apoptosis and inhibit cell invasion andangiogenesis (Bailey et al., 2006). SERPINB5 is both a valuablemolecular marker for the diagnosis and a predictor for the prognosis ofmany cancer types including breast, lung, head and neck, oral andprostate cancer (Marioni et al., 2009; Lonardo et al., 2010; Sager etal., 1996; Sheng, 2004). SERPINB5 acts as an endogenous regulator ofHDAC1 activity and interacts with the p53 tumor suppressor pathway(Maass et al., 2000; Kaplun et al., 2012).

SEZ6L2 encodes a seizure-related protein that is localized on the cellsurface and enriched in pancreatic beta-cells (RefSeq, 2002; Stutzer etal., 2013). The expression of SEZ6L2 is up-regulated in lung cancer anda higher expression level is related to a shorter survival (Ishikawa etal., 2006).

SMARCA4 (also called BRG1) encodes a member of the helicase and ATPasecontaining proteins of the SWI/SNF family that is part of the largeATP-dependent chromatin remodeling complex SWI/SNF. The complex isrequired for transcriptional activation of genes normally repressed bychromatin (RefSeq, 2002). SMARCA4 acts as a tumor suppressor and isdown-regulated via mutations in different cancer entities includingbreast, lung and colon cancer. Low SMARCA4 levels are associated withtumor progression like mutation and invasion (Medina andSanchez-Cespedes, 2008; Bai et al., 2013b; Reisman et al., 2003; Wang etal., 2016). SMARCA4 is related to several tumor suppressors andimportant tumor associated proteins like p53, p16INK4a, hTERT and Akt(Medina and Sanchez-Cespedes, 2008; Becker et al., 2009; Naidu et al.,2009; Liu et al., 2014b; Wu et al., 2014a).

SMC2 (also called CAP-E or SMC2L1) encodes a member of the structuralmaintenance of chromosomes family which is critical for mitoticchromosome condensation and DNA repair (RefSeq, 2002). The SMC2 gene isaltered by frameshift mutation and loss of expression in gastric andcolorectal cancer with microsatellite instability suggesting that SMC2might be involved in tumor pathogenesis (Je et al., 2014). SMC2 genealterations can play a role in genome instability, which accelerates theaccumulation of other alterations in pyothorax-associated lymphomas (Hamet al., 2007).

SVIL encodes supervillin, a bipartite protein with distinct amino- andcarboxy-terminal domains that appears to aid in myosin II assemblyduring cell spreading and disassembly of focal adhesions (RefSeq, 2002).SVIL is significantly down-regulated in prostate cancer tissue mainlythrough promoter methylation (Vanaja et al., 2006). SVIL regulates cellsurvival through control of p53 levels. SVIL expression is necessary forthe cross-talk between survival signaling and cell motility pathways(Fang and Luna, 2013).

TMEM222 encodes the transmembrane protein 222 located on chromosome1p36.11 (RefSeq, 2002).

ZNF679 encodes a zinc finger protein containing a KRAB(Kruppel-associated box) domain that functions as a transcriptionfactor. The promoter region of ZNF679 is bound by the co-repressor KAP1and H3me3K9 (histon 3 trimethylation of lysine 9) (O'Geen et al., 2007).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T-cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13, or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T-cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by aT-cell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 −(1-Gf)2. Combinations of A*02 or A*24 with certain HLA-DR alleles mightbe enriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” shall mean a short nucleic acid sequence that can bepaired with one strand of DNA and provides a free 3′-OH end at which aDNA polymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” shall mean a region of DNA involved in binding ofRNA polymerase to initiate transcription.

The term “isolated” shall mean that the material is removed from itsoriginal environment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 191 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 191, or a variant thereof thatwill induce T-cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T-cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of a given amino acid sequence, the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 191. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T-cells.

These T-cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 191, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T-cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 7, 9,31, 192, 212, and 142 Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 7 Y L T R HL A V L Variants V I A M V M I M M A A V A I A A A V V V I V V A T V T IT T A Q V Q I Q Q A SEQ ID NO. 9 I L D D H L S R V Variants I L A M M IM L M A A A I A L A A V V I V L V A T T I T L T A Q Q I Q L Q A SEQ IDNO. 31 L L Y G K Y V S V Variants I L A M M I M L M A A A I A L A A V VI V L SEQ ID 192

Variants

A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A SEQ ID 212 F L LD G S A N V Variants I L A M M I M L M A A A I A L A A V V I V L V A T TI T L T A Q Q I Q L Q A SEQ ID 142 S L V Q R V E T I Variants V L A M VM M L M A A V A A L A A V V V V L V A T V T T L T A Q V Q Q L Q A

indicates data missing or illegible when filed

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T-cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T-cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 191.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 191 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from CRC samples (N=24 A*02-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 24 CRC patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from CRC tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary CRC samples confirming theirpresentation on primary CRC.

TUMAPs identified on multiple CRC and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Furthermore, the discovery pipeline XPRESIDENT® v2.x allows the directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 12).

The present invention provides peptides that are useful in treatingcancers/tumors, preferably CRC that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanCRC samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy cells from the large intestine (colon or rectum) or other normaltissue cells, demonstrating a high degree of tumor association of thesource genes (see Example 2). Moreover, the peptides themselves arestrongly over-presented on tumor tissue—“tumor tissue” in relation tothis invention shall mean a sample from a patient suffering from CRC,but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T-cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. CRC cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T-cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to the peptides according to the invention can be enhancedby methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluo-rescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription sys-tems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters, TCRexpression cassettes of the present description may contain additionalelements that can enhance transgene expression, including a centralpolypurine tract (cPPT), which promotes the nuclear translocation oflentiviral constructs (Follenzi et al., 2000), and the woodchuckhepatitis virus posttranscriptional regulatory element (wPRE), whichincreases the level of transgene expression by increasing RNA stability(Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3 (CD3 fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH₂ group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin 2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T-cells. However, stimulation ofCD8 T-cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8T-cells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T-cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 191, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill inthe art.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T-cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T-cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T-cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomateous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T-cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T-cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualisation of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 191,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 191, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 191 or a variant thereof thatinduces T-cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:191 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 191, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 191.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain 00, or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of CRC.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 191 or said variant amino acid sequence.

The present invention further relates to activated T-cells, produced bythe method according to the present invention, wherein said T-cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T-cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are CRC cells or other solid orhaematological tumor cells such as lung cancer, brain cancer, stomachcancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovariancancer, and esophageal cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of CRC. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a CRC marker (poly)peptide,delivery of a toxin to a CRC cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a CRC markerpolypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length CRC marker polypeptides or fragments thereof maybe used to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 191polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the CRC marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields an F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed.

For example, it has been described that the homozygous deletion of theantibody heavy chain joining region gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. Human antibodies can also be producedin phage display libraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating CRC, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT-cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴O, ¹³¹I, ³H, ³²P or ³⁵S)) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides.

Additionally, probes may be bi- or multi-functional and be detectable bymore than one of the methods listed. These antibodies may be directly orindirectly labeled with said probes. Attachment of probes to theantibodies includes covalent attachment of the probe, incorporation ofthe probe into the antibody, and the covalent attachment of a chelatingcompound for binding of probe, amongst others well recognized in theart. For immunohistochemistry, the disease tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin. The fixed or embedded section contains the sample arecontacted with a labeled primary antibody and secondary antibody,wherein the antibody is used to detect the expression of the proteins insitu.

Another aspect of the present invention includes an in vitro method forproducing activated T-cells, the method comprising contacting in vitroT-cells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T-cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably, a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T-cellsare CD8-positive T-cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 191, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T-cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T-cells. Furthermore, the production of autologous T-cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T-cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T-cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T-cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T-cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T-cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e.g. cytokines,like interleukin 12.

Allogeneic cells may also be used in the preparation of T-cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T-cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T-cells obtainable by the foregoing methods of theinvention.

Activated T-cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 191.

Preferably, the T-cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T-cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T-cells. The T-cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T-cells). Alternatively, the T-cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T-cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T-cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T-cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T-cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T-cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T-cell. Thisengineered T-cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T-cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from two to sixadministrations) of the reconstituted formulation. The kit may furthercomprise a second container comprising a suitable diluent (e.g., sodiumbicarbonate solution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from CRC, themedicament of the invention is preferably used to treat CRC.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T-cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of CRCpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several CRCtissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, CRC samples from patients andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (CRC)compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T-cells from healthy donors as well as fromCRC patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T-cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from CRC cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for CRC. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T-cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A to 1M show the over-presentation of various peptides in normaltissues (white bars) and CRC (black bars). FIG. 1A: Gene symbol(s):ZNF679, ZNF716, SAPCD2, Peptide: ALIKQLFEA (SEQ ID NO.: 1), Tissues fromleft to right: 1 adipose tissues, 3 adrenal glands, 6 arteries, 3 bonemarrows, 7 brains, 3 breasts, 1 nerv, 1 ovary, 8 esophagi, 2gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 3 lymph nodes,4 leukocyte samples, 3 ovaries, 7 pancreas, 4 peripheral nerves, 1peritoneum, 1 pituitary gland, 2 placentas, 3 pleuras, 1 prostate, 2salivary glands, 4 skeletal muscles, 4 skins, 2 small intestines, 4spleens, 7 stomachs, 4 testes, 2 thymi, 3 thyroid glands, 1 trachea, 1ureter, 3 urinary bladders, 2 uteri, 2 veins, 13 colons, 6 recti, 24CRC. The peptide has additionally been detected on 9/99 lung cancers,2/28 brain cancers, 4/20 ovarian cancers, 1/45 stomach cancers, 1/33prostate cancers, and 2/15 esophageal cancers (not shown). FIG. 1B: Genesymbol(s): BRCA2, Peptide: KQFEGTVEI (SEQ ID NO.: 138), Tissues fromleft to right: 1 adipose tissues, 3 adrenal glands, 6 arteries, 3 bonemarrows, 7 brains, 3 breasts, 1 nerv, 1 ovary, 8 esophagi, 2gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 3 lymph nodes,4 leukocyte samples, 3 ovaries, 7 pancreas, 4 peripheral nerves, 1peritoneum, 1 pituitary gland, 2 placentas, 3 pleuras, 1 prostate, 2salivary glands, 4 skeletal muscles, 4 skins, 2 small intestines, 4spleens, 7 stomachs, 4 testes, 2 thymi, 3 thyroid glands, 1 trachea, 1ureter, 3 urinary bladders, 2 uteri, 2 veins, 13 colons, 6 recti, 24CRC. The peptide has additionally been detected on 1/15 esophagealcancers, 1/28 brain cancers, 1/45 stomach cancers, and 3/91 lung cancers(not shown). FIG. 1C: Gene symbol(s): IL8, Peptide: KLAVALLAA (SEQ IDNO.: 210), Tissues from left to right: 1 adipose tissues, 3 adrenalglands, 6 arteries, 3 bone marrows, 7 brains, 3 breasts, 1 nerv, 1ovary, 8 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 7 pancreas, 4peripheral nerves, 1 peritoneum, 1 pituitary gland, 2 placentas, 3pleuras, 1 prostate, 2 salivary glands, 4 skeletal muscles, 4 skins, 2small intestines, 4 spleens, 7 stomachs, 4 testes, 2 thymi, 3 thyroidglands, 1 trachea, 1 ureter, 3 urinary bladders, 2 uteri, 2 veins, 13colons, 6 recti, 24 CRC. The peptide has additionally been detected on14/99 lung cancers, 1/18 kidney cancers, 2/28 brain cancers, 2/16 livercancers, 1/20 ovarian cancers, 1/45 stomach cancers, and 3/15 esophagealcancers (not shown). FIG. 1D) Gene symbol(s): TMEM222, Peptide:LLYGKYVSV (SEQ ID NO.: 31) Tissues from left to right: 3 pancreatic celllines, 3 skin cell lines, 1 leucocytic cell line, 0 normal tissues, 28cancer tissues (2 brain cancers, 1 breast cancer, 1 colon cancer, 1esophageal cancer, 2 kidney cancers, 1 leukemia, 5 liver cancers, 7 lungcancers, 5 ovarian cancers, 1 prostate cancer, 2 rectal cancers). Thenormal tissue panel tested was the same as in FIGS. 1A-1C. Discrepanciesregarding the list of tumor types between FIG. 1D and table 4 might bedue to the more stringent selection criteria applied in table 4 (fordetails please refer to table 4). FIG. 1D shows all samples withdetectable presentation of the peptide Y, regardless ofover-presentation parameters and technical sample quality check. FIG.1E: Gene symbol(s): ZNF679, ZNF716, SAPCD2, Peptide: ALIKQLFEA (SEQ IDNO.: 1), Tissues from left to right: 7 cancer cell lines, 1 primarycancer cell culture, 58 cancer tissues (5 brain cancers, 1 breastcancer, 9 colon cancers, 1 colorectal cancer, 3 esophageal cancers, 1gallbladder cancer, 2 leukocytic leukemia cancers, 15 lung cancers, 2lymph node cancers, 1 myeloid cells cancer, 5 ovarian cancers, 1prostate cancer, 4 rectum cancers, 1 skin cancer, 2 stomach cancers, 2urinary bladder cancers, 3 uterus cancers). The normal tissue paneltested was the same as in FIG. 1A-C. FIG. 1F: F) Gene symbol(s): PLAGL2,Peptide: FLAELPGSLSL (SEQ ID NO.: 6), Tissues from left to right: 8cancer cell lines, 1 primary cancer cell culture, 2 normal tissues (1lymph node, 1 spleen), 57 cancer tissues (1 bone marrow cancer, 1 breastcancer, 1 cecum cancer, 5 colon cancers, 2 esophageal cancers, 1gallbladder cancer, 3 leukocytic leukemia cancers, 2 liver cancers, 13lung cancers, 8 lymph node cancers, 1 myeloid cells cancer, 9 ovariancancers, 2 rectum cancers, 1 skin cancer, 1 stomach cancer, 4 urinarybladder cancers, 2 uterus cancers). The normal tissue panel tested wasthe same as in FIGS. 1A-1C. FIG. 1G: Gene symbol(s): CYP2W1, Peptide:FLDANGHFV (SEQ ID NO.: 23), Tissues from left to right: 1 primary cancercell culture, 3 normal tissues (3 placentas), 12 cancer tissues (5 coloncancers, 1 esophageal cancer, 1 gallbladder cancer, 2 rectum cancers, 3stomach cancers). The normal tissue panel tested was the same as inFIGS. 1A-1C. FIG. 1H: Gene symbol(s): CYP2W1, Peptide: GLIDEVMVL (SEQ IDNO.: 22), Tissues from left to right: 1 normal tissue (1 stomach), 6cancer tissues (3 colon cancers, 1 gallbladder cancer, 2 rectumcancers). The normal tissue panel tested was the same as in FIGS. 1A-1C.FIG. 1I: Gene symbol(s): AXIN2, Peptide: ILDDHLSRV (SEQ ID NO.: 9),Tissues from left to right: 5 cancer tissues (1 cecum cancer, 1 coloncancer, 1 lung cancer, 2 rectum cancers). The normal tissue panel testedwas the same as in FIGS. 1A-1C. FIG. 1J: Gene symbol(s): RAD54B,Peptide: KLLAVIHEL (SEQ ID NO.: 152), Tissues from left to right: 3 celllines, 2 normal tissues (1 lymph node, 1 spleen), 34 cancer tissues (1breast cancer, 7 colon cancers, 1 esophageal cancer, 1 gallbladdercancer, 1 kidney cancer, 8 lung cancers, 4 lymph node cancers, 1 myeloidcells cancer, 4 ovarian cancers, 1 pancreas cancer, 1 rectum cancer, 3skin cancers, 1 urinary bladder cancer). The normal tissue panel testedwas the same as in FIGS. 1A-1C. FIG. 1K: Gene symbol(s): ECT2, Peptide:SLVQRVETI (SEQ ID NO.: 142), Tissues from left to right: 5 cell lines, 1primary culture, 47 cancer tissues (2 bile duct cancers, 2 breastcancers, 1 cecum cancer, 7 colon cancers, 3 esophageal cancers, 3gallbladder cancers, 1 kidney cancer, 2 liver cancers, 10 lung cancers,2 lymph node cancers, 4 ovarian cancers, 1 pancreas cancer, 2 rectumcancers, 2 skin cancers, 1 stomach cancer, 2 urinary bladder cancers, 2uterus cancers). The normal tissue panel tested was the same as in FIGS.1A-1C. FIG. 1L: Gene symbol(s): MMP12, Peptide: KIQEMQHFL (SEQ ID NO.:192), Tissues from left to right: 1 primary culture, 44 cancer tissues(5 colon cancers, 1 esophageal cancer, 1 gallbladder cancer, 1 head andneck cancer, 30 lung cancers, 1 lymph node cancer, 1 rectum cancer, 1stomach cancer, 1 testis cancer, 1 urinary bladder cancer, 1 uteruscancer). The normal tissue panel tested was the same as in FIGS. 1A-1C.FIG. 1M: Gene symbol(s): COL6A3, Peptide: FLLDGSANV (SEQ ID NO.: 212),Tissues from left to right: 3 cell lines, 2 normal tissues (1 placenta,1 spleen), 146 cancer tissues (4 bile duct cancers, 13 breast cancers, 1cecum cancer, 8 colon cancers, 1 colorectal cancer, 6 esophagealcancers, 5 gallbladder cancers, 5 head and neck cancers, 2 kidneycancers, 1 liver cancer, 62 lung cancers, 2 lymph node cancers, 9ovarian cancers, 7 pancreas cancers, 3 rectum cancers, 4 skin cancers, 5stomach cancers, 5 urinary bladder cancers, 3 uterus cancers). Thenormal tissue panel tested was the same as in FIGS. 1A-1C.

FIGS. 2A to 2C show exemplary expression profiles (relative expressioncompared to normal colon and rectum) of source genes of the presentinvention that are highly over-expressed or exclusively expressed in CRCin a panel of normal tissues (white bars) and 10 CRC samples (blackbars). Tissues from left to right: adrenal gland, artery, bone marrow,brain (whole), breast, colon, esophagus, heart, kidney (triplicate),leukocytes, liver, lung, lymph node, ovary, pancreas, placenta,prostate, salivary gland, skeletal muscle, skin, small intestine,spleen, stomach, testis, thymus, thyroid gland, urinary bladder, uterinecervix, uterus, vein, 3 normal colon samples, 10 CRC samples. FIG. 2A,CCNB1; FIG. 2B, CDK1; FIG. 2C, CHMP5. FIG. 2D shows exemplary expressionprofiles (relative expression compared to normal colon and rectum) ofsource genes of the present invention that are highly over-expressed orexclusively expressed in CRC in a panel of normal tissues (white bars)and 20 CRC samples (black bars). Tissues from left to right: 6 arteries,2 blood cells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adiposetissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1esophagus, 2 eyes, 2 gallbladders, 2 salivary glands, 1 kidney, 6 lymphnodes, 4 pancreases, 2 peripheral nerves, 2 pituitary glands, 1 rectum,2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 5placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus, 20 CRC samples.FIG. 2D: ECT2.

FIG. 3 shows exemplary immunogenicity data: flow cytometry results afterpeptide-specific multimer staining.

FIGS. 4A to 4C show exemplary results of peptide-specific in vitro CD8+T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complexwith SeqID No 22 peptide (FIG. 4A, left panel), SeqID No 9 peptide (FIG.4B, left panel) or SeqID No 142 peptide (FIG. 4C, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/SeqID No 22 (FIG. 4A), A*02/SeqID No 9 (FIG. 4B) or A*02/SeqID No142 (FIG. 4C). Right panels (FIGS. 4A, 4B and 4C) show control stainingof cells stimulated with irrelevant A*02/peptide complexes. Viablesinglet cells were gated for CD8+ lymphocytes. Boolean gates helpedexcluding false-positive events detected with multimers specific fordifferent peptides. Frequencies of specific multimer+ cells among CD8+lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from University Hospital ofTübingen.

Normal tissues were obtained from Asterand, Detroid, USA and Royston,Herts, UK; Bio-Options Inc, CA, USA; BioServe, Beltsville, Md., USA;Capital BioScience Inc, Rockville, Md., USA; Geneticist Inc., Glendale,Calif., USA; Tissue Solutions Ltd, Glasgow, Scotland, UK; UniversityHospital of Geneva; University Hospital of Heidelberg; Kyoto PrefecturalUniversity of Medicine (KPUM); University Hospital Munich; ProteoGenexInc., Culver City, Calif., USA; University Hospital of Tübingen. Writteninformed consents of all patients had been given before surgery orautopsy. Tissues were shock-frozen immediately after excision and storeduntil isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose CRC samples to a baseline of normal tissue samples.Presentation profiles of exemplary over-presented peptides are shown inFIG. 1A-1M. Presentation scores for exemplary peptides are shown inTable 8.

TABLE 8 Presentation scores. The table lists peptidesthat are very highly over-presented on tumorscompared to a panel of normal tissues (+++),highly over-presented on tumors compared toa panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+). SEQ IDPeptide No. Sequence Presentation   1 ALIKQLFEA +++   2 ALLPRYFFL +++  3 RLIPDTLYSV +++   4 RLAELTVDEFL +++   5 WLFDDGGLTL +++   6FLAELPGSLSL +   7 YLTRHLAVL +++   8 ALMLQGVDLL +++   9 ILDDHLSRV +++  10RMYNKIFAI +++  11 YLFEKTFNM +++  12 ALVQGILERV +++  13 FLLAEDTKV +++  15LQLDKEFQL +  16 VLVDQSVVVL +++  17 ALAAARVEL +++  18 FLSSLKGGLL +++  19RLYTKLLNEA +++  21 VLIDHRVVVL +++  22 GLIDEVMVL +++  23 FLDANGHFV +  25SLADRLIGV +++  26 GLASKENFSNVSL +++  27 LLADEDSSYL +++  30 GLSSAYGGL +++ 31 LLYGKYVSV +++  32 KLNTETFGV +++  33 ALWEKNTHL +++  34 ILLEKSVSV +++ 35 KLLDLTVRI +++  36 GLLESPSIFNFTA +++  37 GLFAGLGGAGA +++  38SLAPTPVSA +++  39 GLNGGSPAAA +++  40 ALSNVIHKV +++  41 ILDDSFKLL ++  42SILDDSFKL +++  43 TLDAAQPRV ++  44 SLESKLTSV +++  45 ALAELLHGA +++  46GLDDRYSLV +  47 KLYERCEVV ++  48 FLDASDPAL +++  51 QVWEIQHTV ++  53FLLGSEIKL ++  54 ALLNGEYLLAA +  56 VLFTDEGVPKFL +  57 NLLEKENYL ++  58AMADKMDMSL +  59 LLTDNVVKL +  60 VLDEDEPRFL +  61 KLLKLFQGV +++  62YLAPENGYL ++  63 KLFSILSTV +  64 KTLGKLWRL +++  65 FGAPGIISA +++  66GLDDGPDFL +  67 SLNDLEKDVMLL +  68 SILQFVHMV ++  69 GMLNEAEGKAIKL +  70MISELEVRL +  71 RLWTEIPTAI ++  72 YLLDYPNNLL ++  73 YLFDIAVSM ++  74YLMGFLHAV ++  75 EMIENIQSV +  77 SLLKRDFGA +  78 ALDPELLLL +  80QVDEVVDIMRV ++  81 ALLSQQTHL ++  82 QLYEEPDTKL ++  83 LTIEDGIFEV +  88KLDIKVETV +  89 SLIEYEFRV ++  90 GLLKPGLNVVL +  92 WIDDTSAFV +++  93SLQELRLLL +  95 AILDAHIEV +  96 KLYSRLVYV ++  97 ALVWVGVVTV ++ 100SLDDFLATA + 102 KILVSLIEV +++ 103 FLFGYPKRL + 110 LLGELPRLLLL + 111HMDDGGYSM + 112 KLGQVLIYL +++ 113 ILYDLQQNL + 123 KTLERSYLL +++ 124RVLPPSALQSV ++ 125 KLGDFGLLVEL +++ 126 TLAKYLMEL +++ 127 RLAELTVDEFLA+++ 128 MLDDRAYLV ++ 129 VLIDVLKEL +++ 130 GLGGSQLIDTHL +++ 131KLLDVVHPA +++ 132 ALLNAILHSA +++ 133 RTFEKIEEV +++ 134 GVAGGSILKGV +++135 KLQEEIPVL +++ 136 KLFDIFSQQV +++ 137 QLTEIKPLL +++ 138 KQFEGTVEI +++139 VLLNEILEQV + 141 AVIEHLERL +++ 142 SLVQRVETI +++ 143 KLSDVWKEL +++144 LLNDRIWLA + 145 LLLEVVKQV +++ 146 ALSDETWGL + 148 RLLENMTEVV ++ 150RLADLEALKV +++ 152 KLLAVIHEL + 153 ILFSEDSTKLFV + 154 KLPSETIFVGC + 155RLLGEEVVRV ++ 156 SLMMTIINL ++ 157 SLIERDLKL ++ 158 GLLDPSVFHV +++ 159VLVDDDGIKVV +++ 160 KLLEFDQLQL ++ 161 FLKNELDNV ++ 162 KLMDYIDEL ++ 163RLLHEVQEL ++ 164 KMLDEILLQL ++ 165 RLLDFPEAMVL +++ 166 GLLEARGILGL + 168GLIRFPLMTI ++ 170 ALAGGITMV ++ 171 RLQETEGMVAV + 172 LLLDTVTMQV + 173KLGDLMVLL + 177 ALLQGAIESV + 178 YLFREPATI + 179 RLLJPLSSA + 180NLLEIAPHL ++ 183 TLQEVVTGV + 185 VLYTGVVRV + 186 KMSEKILLL + 187GLHNVVYGI ++ 188 FLVDGPRVQL + 192 KIQEMQHFL +++ 193 KLSPTVVGL +++ 194SLYKGLLSV +++ 195 LLLGERVAL +++ 198 VLYGPDVPTI ++ 199 FLLEREQLL +++ 201GJFNGALAAV +++ 202 GLAALAVHL +++ 203 KLIDLSQVMYL + 204 KLLDLETERILL ++205 RLHDENILL +++ 206 RIAGIRGIQGV ++ 207 KLCEGFNEV +++ 208 RLIDRIKTV +++209 KLQDGLLHI +++ 210 KLAVALLAA +++ 211 SLFGKKYIL +++ 213 LLWAPTAQA +++214 SVLEKEIYSI +++ 215 KLQEKIQEL +++ 216 YLWDLDHGFAGV +++ 217KLLDTMVDTFL ++ 218 KLSWDLIYL + 220 KMDPVAYRV + 221 ILNVDGLIGV + 223VLMQDSRLYL +++ 224 QLQEGKNVIGL +++ 225 YLYGQTTTYL + 226 FLVDGSWSV + 227LTAPPEALLMV ++ 228 SMSGYDQVL + 229 YLLEKFVAV ++ 230 AMSSKFFLV ++ 231RLFADILNDV +++ 232 RLLDSVSRL + 233 RLDDLKMTV ++ 234 KMFESFIESV ++ 235LLHEENFSV ++ 236 KMSELQTYV + 237 KLVEFDFLGA ++ 238 NMLEAVHTI ++ 239QLIEKNWLL +++ 240 VLAPRVLRA ++ 241 ILIDWLVQV + 242 RLEEDDGDVAM ++ 243TLMDMRLSQV + 244 SLHFLILYV + 245 QLIDYERQL + 246 GLTDNIHLV + 247SLDTLMTYV + 249 ALYGRLEVV + 250 ALCEENMRGV + 252 YVYQNNIYL + 254VLFQEALWHV ++ 257 SLADFMQEV ++ 259 ALADKELLPSV + 261 YLYDSETKNA +

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed in CRCare shown in FIGS. 2A-2D. Expression scores for further exemplary genesare shown in Table 9.

TABLE 9 Expression scores. The table lists peptides from genes that are very highly over-expressed in tumors compared to a panel ofnormal tissues (+++), highly over-expressed in tumors compared to a panel of normaltissues (++) or over-expressed in tumorscompared to a panel of normal tissues (+). SEQ ID Gene NO. Gene NameSequence Expression   2 ATP10B ALLPRYFFL +++   5 SLC12A1, WLFDDGGLTL +++SLC12A2, SLC12A3   6 PLAGL2 FLAELPGSLSL +++   7 MUC2 YLTRHLAVL +   8HSPD1 ALMLQGVDLL +  13 SMC2 FLLAEDTKV +++  16 KLK10 VLVDQSVVVL +  17SLC12A2 ALAAARVEL +++  19 MYO10 RLYTKLLNEA +++  27 CHMP5 LLADEDSSYL ++ 29 AP3D1 QMLDVAIRV +  35 OLFM4 KLLDLTVRI +  36 LARP4B GLLESPSIFNFTA + 39 CDX2 GLNGGSPAAA ++  40 SERPINB5 ALSNVIHKV +  41 HEPH ILDDSFKLL ++ 42 HEPH SILDDSFKL ++  46 PKP3 GLDDRYSLV +  47 ERBB3 KLYERCEVV +  53TBC1D8B FLLGSEIKL +  55 PMS1 QIITSVVSV ++  57 PKP2 NLLEKENYL ++  60AGTPBP1 VLDEDEPRFL +  63 HEATR2 KLFSILSTV ++  64 SOX8, KTLGKLWRL ++SOX9, SOX10  67 SMARCA4 SLNDLEKDVMLL ++  68 PTPRO SILQFVHMV +  73 APIPYLFDIAVSM +  74 ARHGAP8, YLMGFLHAV + PRR5- ARHGAP8, PRR5  75 CFTREMIENIQSV +++  77 DDX5 SLLKRDFGA +  79 SRSF11 SLAADQLLKL ++  81 TGIF1ALLSQQTHL +  84 DSP SMVEDITGLRL +  86 MUC13 KVFPGKISV +++  89 ITGA6SLIEYEFRV ++  90 EBNA1BP2 GLLKPGLNVVL ++  92 PARN WIDDTSAFV +  98ATP13A3 AMNGKSFSV +++ 104 MUC2 ILLTIKDDTIYL + 112 GALNT7 KLGQVLIYL ++113 KCNN4 ILYDLQQNL + 123 RRM1 KTLERSYLL +++ 124 AURKB RVLPPSALQSV ++126 CCNB1, TLAKYLMEL +++ CCNB2 129 CNOT1 VLIDVLKEL + 130 PRRC2CGLGGSQLIDTHL ++ 132 NOL11 ALLNAILHSA ++ 134 EIF2S3, GVAGGSILKGV +LOC255308 135 CENPE KLQEEIPVL + 138 BRCA2 KQFEGTVEI ++ 139 NCAPGVLLNEILEQV +++ 140 NCAPG LLNEILEQV +++ 142 ECT2 SLVQRVETI ++ 144 ZSWIM1LLNDRIWLA ++ 147 KDM5C TLTELRAFL + 148 PDXDC1 RLLENMTEVV + 152 RAD54BKLLAVIHEL + 156 TOP2A SLMMTIINL +++ 157 URB1 SLIERDLKL + 160 SYNJ2KLLEFDQLQL + 161 TRAIP FLKNELDNV + 166 CDC6 GLLEARGILGL + 171 HMGXB4RLQETEGMVAV + 172 COPG1 LLLDTVTMQV + 180 GPD2 NLLEIAPHL + 183 AGKTLQEVVTGV ++ 184 PRKDC SLLDENNVSSYL + 187 CNOT1 GLHNVVYGI + 188 ZSWIM1FLVDGPRVQL ++ 190 NCAPD2 AMAEMVLQV + 191 CDK5RAP2 QLFSEIHNL + 192 MMP12KIQEMQHFL ++ 194 RAD54B SLYKGLLSV + 197 ZNF451 SLFGQDVKAV + 198 CEACAM6VLYGPDVPTI ++ 202 FANCA GLAALAVHL ++ 204 GOLGA4 KLLDLETERILL + 205RPGRIP1L RLHDENILL + 206 EFR3A RIAGIRGIQGV + 208 NAA35 RLIDRIKTV + 215CENPE KLQEKIQEL + 219 MUC2 FLDEKGRCV + 223 CDK1 VLMQDSRLYL +++ 225 TOP2AYLYGQTTTYL +++ 228 HNRNPH1, SMSGYDQVL +++ HNRNPH2 229 DDX11, YLLEKFVAV +DDX12P, LOC642846 + 230 WNT5A AMSSKFFLV + 232 LAMC2 RLLDSVSRL ++ 233LAMC2 RLDDLKMTV ++ 235 TCF20 LLHEENFSV + 236 CENPF KMSELQTYV ++ 239KIF15 QLIEKNWLL +++ 240 RCN1 VLAPRVLRA ++ 241 CCNB1 ILIDWLVQV +++ 250EEF2 ALCEENMRGV + 257 CNOT1 SLADFMQEV +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T-cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 22 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T-cells exist in humans (Table10).

In Vitro Priming of CD8+ T-Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T-cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nûrnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 266) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.267), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T-cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by by comparing to negativecontrol stimulations. Immunogenicity for a given antigen was detected ifat least one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for CRC Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 1peptide of the invention are shown in FIG. 3 together with correspondingnegative controls. Results for 2 peptides from the invention aresummarized in Table 10A.

TABLE 10A in vitro immunogenicity of HLA class I peptides of theinvention Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for the peptides of the invention. Seq IDPeptide ID wells donors 219 MUC2-001 ++ +++ 220 QAR-001 +++ ++++ <20% =+; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

TABLE 10B Additional data for in vitro immunogenicityof HLA class I peptides of the invention.Exemplary results of in vitro immunogenicityexperiments conducted by the applicant forHLA-A*02 restricted peptides of the invention.Results of in vitro immunogenicity experimentsare indicated. Percentage of positive wells anddonors (among evaluable) are summarized as indicated <20% = +; 20%-49% =++; 50%-69% = +++; >=70% = ++++ SEQ Wells ID positive NO Sequence [%]  1 ALIKQLFEA “+”   2 ALLPRYFFL “++++”   3 RLIPDTLYSV “+++”   5WLFDDGGLTL “++”   7 YLTRHLAVL “+”   9 ILDDHLSRV “+”  10 RMYNKIFAI “++++” 11 YLFEKTFNM “+”  12 ALVQGILERV “++++”  13 FLLAEDTKV “++”  17 ALAAARVEL“++”  18 FLSSLKGGLL “+”  19 RLYTKLLNEA “+++”  21 VLIDHRVVVL “+”  22GLIDEVMVL “++”  31 LLYGKYVSV “++”  32 KLNTETFGV “++”  37 GLFAGLGGAGA “+” 38 SLAPTPVSA “+”  42 SILDDSFKL “+”  47 KLYERCEVV “+”  59 LLTDNVVKL “+” 64 KTLGKLWRL “++++” 123 KTLERSYLL “+” 124 RVLPPSALQSV “+” 127RLAELTVDEFLA “+” 132 ALLNAILHSA “+” 133 RTFEKIEEV “+” 136 KLFDIFSQQV“++” 141 AVIEHLERL “+” 142 SLVQRVETI “+” 150 RLADLEALKV “++”

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO4. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*02:01was ranged by peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50% =+++; ≥75% = ++++; J = Phosphoserine SEQ ID Peptide NO Sequence exchange  1 ALIKQLFEA “+++”   2 ALLPRYFFL “++”   3 RLIPDTLYSV “++++”   4RLAELTVDEFL “+++”   5 WLFDDGGLTL “++++”   6 FLAELPGSLSL “+++”   7YLTRHLAVL “++”   8 ALMLQGVDLL “+++”   9 ILDDHLSRV “++”  10 RMYNKIFAI“+++”  11 YLFEKTFNM “+++”  12 ALVQGILERV “+++”  13 FLLAEDTKV “+++”  14FLDKPEDVLL “++”  15 LQLDKEFQL “+++”  16 VLVDQSVVVL “+++”  17 ALAAARVEL“+++”  18 FLSSLKGGLL “+++”  19 RLYTKLLNEA “+++”  20 YLKDGDVML “+++”  21VLIDHRVVVL “+++”  22 GLIDEVMVL “+++”  23 FLDANGHFV “+++”  24 VLDGVLMEL“+++”  25 SLADRLIGV “++++”  26 GLASKENFSNVSL “++”  27 LLADEDSSYL “++” 28 ALTEIQEFI “++++”  29 QMLDVAIRV “+++”  30 GLSSAYGGL “+”  31 LLYGKYVSV“+++”  32 KLNTETFGV “++”  33 ALWEKNTHL “+++”  34 ILLEKSVSV “+++”  35KLLDLTVRI “+++”  36 GLLESPSIFNFTA “+++”  37 GLFAGLGGAGA “+++”  38SLAPTPVSA “++”  40 ALSNVIHKV “++”  41 ILDDSFKLL “++”  42 SILDDSFKL“++++”  43 TLDAAQPRV “++”  44 SLESKLTSV “+++”  45 ALAELLHGA “+++”  46GLDDRYSLV “+++”  47 KLYERCEVV “++”  48 FLDASDPAL “++”  50 TLMAEMHVV“+++”  51 QVWEIQHTV “++”  52 ALDSSNSMQTI “++”  53 FLLGSEIKL “+++”  54ALLNGEYLLAA “+++”  56 VLFTDEGVPKFL “++”  57 NLLEKENYL “+++”  58AMADKMDMSL “++”  59 LLTDNVVKL “+++”  60 VLDEDEPRFL “++”  61 KLLKLFQGV“+++”  62 YLAPENGYL “++”  63 KLFSILSTV “++”  64 KTLGKLWRL “++”  66GLDDGPDFL “++”  67 SLNDLEKDVMLL “+++”  68 SILQFVHMV “+++”  69GMLNEAEGKAIKL “++”  70 MISELEVRL “+++”  71 RLVVTEIPTAI “+++”  72YLLDYPNNLL “+++”  73 YLFDIAVSM “+++”  74 YLMGFLHAV “+++”  75 EMIENIQSV“++”  76 YLIGEKQHYL “+++”  77 SLLKRDFGA “++”  78 ALDPELLLL “++”  79SLAADQLLKL “++”  80 QVDEVVDIMRV “++”  81 ALLSQQTHL “+++”  82 QLYEEPDTKL“++”  83 LTIEDGIFEV “+++”  84 SMVEDITGLRL “+++”  85 ILHDINSDGVL “++”  86KVFPGKISV “++”  87 LLFDAPDLRL “+++”  88 KLDIKVETV “++++”  89 SLIEYEFRV“+++”  90 GLLKPGLNVVL “+++”  91 TVDVATPSV “+++”  92 WIDDTSAFV “+++”  93SLQELRLLL “++++”  94 KSMDIVLTV “+++”  95 AILDAHIEV “++++”  96 KLYSRLVYV“++”  97 ALVWVGVVTV “++”  98 AMNGKSFSV “++”  99 KLLEVDLDTV “+++” 100SLDDFLATA “+++” 101 GLSEGHTFQV “+++” 102 KILVSLIEV “+++” 103 FLFGYPKRL“++” 104 ILLTIKDDTIYL “+++” 105 YALDLSTFL “+++” 106 SLISEKILL “+++” 107ALLGGGPYML “+++” 108 SLAELVPGVGGI “+++” 109 ALDGDQMEL “++” 110LLGELPRLLLL “+++” 112 KLGQVLIYL “++” 113 ILYDLQQNL “++” 114 TAVGHALVL“+” 115 SLFDVSHML “+++” 116 LVYQFVHPI “++” 117 TLQPVDNSTISL “++” 118LLADLKTMV “+++” 119 ILYQTVTGL “++” 120 VLYEGVDEV “++” 121 SLAPNIISQL“+++” 122 SLMGMVLKL “+++” 123 KTLERSYLL “++” 124 RVLPPSALQSV “+++” 125KLGDFGLLVEL “++++” 126 TLAKYLMEL “+++” 127 RLAELTVDEFLA “+++” 128MLDDRAYLV “++” 129 VLIDVLKEL “+++” 130 GLGGSQLIDTHL “++” 131 KLLDVVHPA“++” 132 ALLNAILHSA “+++” 133 RTFEKIEEV “++” 134 GVAGGSILKGV “++++” 135KLQEEIPVL “++” 136 KLFDIFSQQV “+++” 137 QLTEIKPLL “+++” 138 KQFEGTVEI“+++” 139 VLLNEILEQV “+++” 140 LLNEILEQV “+++” 141 AVIEHLERL “++++” 142SLVQRVETI “+++” 143 KLSDVWKEL “+++” 144 LLNDRIWLA “+++” 145 LLLEVVKQV“+++” 146 ALSDETWGL “++” 147 TLTELRAFL “++++” 148 RLLENMTEVV “+++” 149YQFDKVGILTL “+++” 150 RLADLEALKV “+++” 151 SAQGSDVSLTACKV “+++” 152KLLAVIHEL “++” 153 ILFSEDSTKLFV “+++” 154 KLPSETIFVGC “+++” 155RLLGEEVVRV “+++” 156 SLMMTIINL “++++” 157 SLIERDLKL “+++” 158 GLLDPSVFHV“+++” 159 VLVDDDGIKVV “++” 160 KLLEFDQLQL “+++” 161 FLKNELDNV “+++” 162KLMDYIDEL “+++” 163 RLLHEVQEL “+++” 164 KMLDEILLQL “++++” 165RLLDFPEAMVL “++++” 166 GLLEARGILGL “+++” 167 SVIDHIHLISV “+++” 168GLIRFPLMTI “+++” 169 YLAHFIEGL “+++” 170 ALAGGITMV “+++” 171 RLQETEGMVAV“++” 172 LLLDTVTMQV “+++” 173 KLGDLMVLL “+++” 174 ILLDDNMQIRL “++++” 175TLLGGKEAQALGV “+++” 176 RTLDKVLEV “++” 177 ALLQGAIESV “+++” 178YLFREPATI “++” 179 RLLJPLSSA “+++” 181 NLFDLGGQYLRV “+++” 182 SLNKWIFTV“++++” 183 TLQEVVTGV “+++” 184 SLLDENNVSSYL “+++” 185 VLYTGVVRV “+++”186 KMSEKILLL “+++” 187 GLHNVVYGI “+++” 188 FLVDGPRVQL “+++” 189AISEVIGKITA “+++” 190 AMAEMVLQV “+++” 191 QLFSEIHNL “++++”

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and -specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in EXAMPLE 1, the inventors did analyze absolute peptidecopies per cell as described in patent x. The quantitation of TUMAPcopies per cell in solid tumor samples requires the absolutequantitation of the isolated TUMAP, the efficiency of TUMAP isolation,and the cell count of the tissue sample analyzed. An overview on ourexperimental approach is given in FIGS. 4A-4C, experimental steps aredescribed below.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard; theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides are shownin Table 12

TABLE 12 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in NSCLC tumor samples. The median number of copiesper cell are indicated for each peptide: SEQ ID Copies per cell Numberof No. Peptide Code (median) samples 1 ZNF-002 + 19 142 ECT2-001 + 18 22CYP2W1-001 ++ 23 152 RAD54B-002 +++ 6 <100 = +; >=100 = ++; >=1,000+++; >=10,000 = ++++. The number of samples, in which evaluable, highquality MS data are available, is indicated.

REFERENCE LIST

-   Allison, J. P. et al., Science 270 (1995): 932-933-   Andersen, R. S. et al., Nat. Protoc. 7 (2012): 891-902-   Appay, V. et al., Eur. J Immunol. 36 (2006): 1805-1814-   Banchereau, J. et al., Cell 106 (2001): 271-274-   Beatty, G. et al., J Immunol 166 (2001): 2276-2282-   Beggs, J. D., Nature 275 (1978): 104-109-   Benjamini, Y. et al., Journal of the Royal Statistical Society.    Series B (Methodological), Vol. 57 (1995): 289-300-   Boulter, J. M. et al., Protein Eng 16 (2003): 707-711-   Braumuller, H. et al., Nature (2013)-   Brossart, P. et al., Blood 90 (1997): 1594-1599-   Bruckdorfer, T. et al., Curr. Pharm. Biotechnol. 5 (2004): 29-43-   Card, K. F. et al., Cancer Immunol Immunother. 53 (2004): 345-357-   Chanock, S. J. et al., Hum. Immunol. 65 (2004): 1211-1223-   Cohen, C. J. et al., J Mol Recognit. 16 (2003a): 324-332-   Cohen, C. J. et al., J Immunol 170 (2003b): 4349-4361-   Cohen, S. N. et al., Proc. Natl. Acad. Sci. U.S.A 69 (1972):    2110-2114-   Coligan J E et al., (1995)-   Colombetti, S. et al., J Immunol. 176 (2006): 2730-2738-   Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170-   Denkberg, G. et al., J Immunol 171 (2003): 2197-2207-   Falk, K. et al., Nature 351 (1991): 290-296-   Fong, L. et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001): 8809-8814-   Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103-   Gattinoni, L. et al., Nat Rev. Immunol 6 (2006): 383-393-   Gnjatic, S. et al., Proc Natl. Acad. Sci. U.S.A 100 (2003):    8862-8867-   Godkin, A. et al., Int. Immunol 9 (1997): 905-911-   Green M R et al., 4th, (2012)-   Greenfield E A, 2nd, (2014)-   Hwang, M. L. et al., J Immunol. 179 (2007): 5829-5838-   Jung, G. et al., Proc Natl Acad Sci USA 84 (1987): 4611-4615-   Kibbe A H, rd, (2000)-   Krieg, A. M., Nat Rev. Drug Discov. 5 (2006): 471-484-   Liddy, N. et al., Nat Med. 18 (2012): 980-987-   Ljunggren, H. G. et al., J Exp. Med. 162 (1985): 1745-1759-   Longenecker, B. M. et al., Ann N.Y. Acad. Sci. 690 (1993): 276-291-   Lukas, T. J. et al., Proc. Natl. Acad. Sci. U.S.A 78 (1981):    2791-2795-   Lundblad R L, 3rd, (2004)-   Meziere, C. et al., J Immunol 159 (1997): 3230-3237-   Morgan, R. A. et al., Science 314 (2006): 126-129-   Mori, M. et al., Transplantation 64 (1997): 1017-1027-   Mortara, L. et al., Clin Cancer Res. 12 (2006): 3435-3443-   Mueller, L. N. et al., J Proteome. Res 7 (2008): 51-61-   Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480-   Mumberg, D. et al., Proc. Natl. Acad. Sci. U.S.A 96 (1999):    8633-8638-   Pinheiro J et al., (2015)-   Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787-   Porta, C. et al., Virology 202 (1994): 949-955-   Rammensee, H. G. et al., Immunogenetics 50 (1999): 213-219-   Rini, B. I. et al., Cancer 107 (2006): 67-74-   Rock, K. L. et al., Science 249 (1990): 918-921-   Rodenko, B. et al., Nat Protoc. 1 (2006): 1120-1132-   Saiki, R. K. et al., Science 239 (1988): 487-491-   Seeger, F. H. et al., Immunogenetics 49 (1999): 571-576-   Sherman F et al., (1986)-   Singh-Jasuja, H. et al., Cancer Immunol. Immunother. 53 (2004):    187-195-   Small, E. J. et al., J Clin Oncol. 24 (2006): 3089-3094-   Sturm, M. et al., BMC. Bioinformatics. 9 (2008): 163-   Teufel, R. et al., Cell Mol Life Sci. 62 (2005): 1755-1762-   Tran, E. et al., Science 344 (2014): 641-645-   Walter, S. et al., J Immunol 171 (2003): 4974-4978-   Walter, S. et al., Nat Med. 18 (2012): 1254-1261-   Wilcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423-   Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577-   Albrecht, M. et al., FEBS Lett. 569 (2004): 18-26-   Albulescu, R., Biomark. Med. 7 (2013): 203-   Aschauer, H. et al., Wien. Klin. Wochenschr. 95 (1983): 785-788-   Aung, P. P. et al., Oncogene 25 (2006): 2546-2557-   Backen, A. C. et al., Br. J Cancer 96 (2007): 1544-1548-   Bai, J. et al., PLoS. One. 8 (2013b): e59772-   Bailey, C. M. et al., J Cell Physiol 209 (2006): 617-624-   Baris, O. et al., J Clin Endocrinol. Metab 89 (2004): 994-1005-   Becker, T. M. et al., Mol. Cancer 8 (2009): 4-   Bie, L. et al., PLoS. One. 6 (2011): e25631-   Bilbao-Aldaiturriaga, N. et al., Pediatr. Blood Cancer 62 (2015):    766-769-   Boland, A. et al., Nat Struct. Mol. Biol 20 (2013): 1289-1297-   Bulk, E. et al., Int. J Cancer 137 (2015): 1306-1317-   Cao, R. et al., Br. J Cancer 111 (2014): 539-550-   Carvalho, L. et al., Rev Port. Pneumol. 15 (2009): 683-696-   Chang, H. Y. et al., PLoS. One. 8 (2013): e54117-   Chen, C. H. et al., Oncotarget. 5 (2014a): 6300-6311-   Chen, Y. D. et al., Zhonghua Lao. Dong. Wei Sheng Zhi. Ye. Bing. Za    Zhi. 30 (2012): 725-729-   Chou, C. C. et al., Expert. Rev Mol. Diagn. 8 (2008): 179-187-   Chung, F. Y. et al., J Surg. Oncol 102 (2010): 148-153-   Cohen, Y. et al., Hematology. 19 (2014): 286-292-   Cole, C. L. et al., J Biol Chem 289 (2014): 10488-10501-   Courson, D. S. et al., Exp. Cell Res 334 (2015): 10-15-   Di, Maro G. et al., J Clin Endocrinol. Metab 99 (2014): E1617-E1626-   Ding, K. et al., Med. Hypotheses 83 (2014): 359-364-   Doherty, J. A. et al., Cancer Epidemiol. Biomarkers Prev. 20 (2011):    1873-1882-   Duursma, A. et al., Mol. Cell Biol 25 (2005): 6937-6947-   Egloff, A. M. et al., Cancer Res 66 (2006): 6-9-   Fan, C. G. et al., Oncol Rep. 26 (2011): 1281-1286-   Fang, Y. et al., Cancer Biol Ther. 15 (2014): 1268-1279-   Fang, Z. et al., J Biol Chem 288 (2013): 7918-7929-   Fang, Z. Q. et al., Genet. Mol Res 12 (2013): 1479-1489-   Feng, B. et al., J Gastroenterol. Hepatol. 21 (2006): 1596-1603-   Ferreras, C. et al., J Biol Chem 287 (2012): 36132-36146-   Fields, A. P. et al., Adv. Enzyme Regul. 50 (2010): 190-200-   Flanagan, J. M. et al., Mol. Cancer Ther. 8 (2009): 249-260-   Freed, E. F. et al., PLoS. Genet. 8 (2012): e1002892-   Goldenson, B. et al., Oncogene 34 (2015): 537-545-   Gomez, A. et al., Mol. Pharmacol. 78 (2010): 1004-1011-   Griffin, J. N. et al., PLoS. Genet. 11 (2015): e1005018-   Gutierrez-Camino, A. et al., Pediatr. Res 75 (2014): 767-773-   Ham, M. F. et al., Cancer Sci. 98 (2007): 1041-1047-   Hanks, T. S. et al., Apoptosis. 17 (2012): 236-247-   Haren, N. et al., Histol. Histopathol. 25 (2010): 1247-1255-   Hatabe, S. et al., Mol. Clin Oncol 1 (2013): 845-850-   Hayama, S. et al., Cancer Res 67 (2007): 4113-4122-   Hegyi, K. et al., Pathobiology 79 (2012): 314-322-   Hill, S. J. et al., Genes Dev. 28 (2014): 1957-1975-   Hiramoto, T. et al., Oncogene 18 (1999): 3422-3426-   Hu, J. et al., Pituitary. 10 (2007): 47-52-   Hu, S. X. et al., Zhonghua Lao. Dong. Wei Sheng Zhi. Ye. Bing. Za    Zhi. 31 (2013): 890-894-   Huff, L. P. et al., Genes Cancer 4 (2013): 460-475-   Ishikawa, N. et al., Cancer Sci. 97 (2006): 737-745-   Ito, K. et al., Protein Cell 2 (2011): 755-763-   Jager, D. et al., Cancer Res 60 (2000): 3584-3591-   Januchowski, R. et al., Biomed. Res Int 2014 (2014): 365867-   Jin, Y. et al., Int. J Clin Exp. Pathol. 7 (2014): 8724-8731-   Jordheim, L. P. et al., Biomark. Med. 7 (2013): 663-671-   Jordheim, L. P. et al., Lancet Oncol 12 (2011): 693-702-   Ju, W. et al., Oncol. Res. 18 (2009): 47-56-   Kanda, A. et al., Oncogene 24 (2005): 7266-7272-   Kaplun, A. et al., Crit Rev Eukaryot. Gene Expr. 22 (2012): 249-258-   Karlgren, M. et al., Expert. Opin. Ther. Targets. 11 (2007): 61-67-   Kas, K. et al., J Biol Chem 273 (1998): 23026-23032-   Kaur, S. et al., BMC. Cell Biol 9 (2008): 61-   Kim, D. H., Yonsei Med. J 48 (2007): 694-700-   Kim, D. S. et al., J Proteome. Res 9 (2010a): 3710-3719-   Kim, J. E. et al., J Cancer Res Clin Oncol 136 (2010b): 47-53-   Kounelakis, M. G. et al., IEEE J Biomed. Health Inform. 17 (2013):    128-135-   Lages, E. et al., PLoS. One. 6 (2011): e20600-   Lallet-Daher, H. et al., Oncogene 28 (2009): 1792-1806-   Landrette, S. F. et al., Blood 105 (2005): 2900-2907-   Langnaese, K. et al., Cytogenet. Cell Genet. 94 (2001): 233-240-   Lee, Y. C. et al., Int. J Cancer 122 (2008b): 1630-1638-   Li, B. et al., Cancer Res 61 (2001): 8014-8021-   Li, G. H. et al., Bioinformatics. 30 (2014): 748-752-   Li, J. F. et al., Zhonghua Wei Chang Wai Ke. Za Zhi. 15 (2012):    388-391-   Li, Y. et al., PLoS. One. 8 (2013): e84489-   Liu, B. et al., Int. J Clin Exp. Pathol. 7 (2014a): 3089-3100-   Liu, L. et al., Retrovirology. 8 (2011a): 94-   Liu, X. et al., Eur. J Cancer 50 (2014b): 2251-2262-   Liu, Y. et al., Cancer Epidemiol. Biomarkers Prev. 18 (2009):    204-214-   Liu, Y. et al., J Cancer 6 (2015b): 643-651-   Lonardo, F. et al., Curr. Pharm. Des 16 (2010): 1877-1881-   Lu, X. et al., Mol. Cancer Ther. 3 (2004): 861-872-   Maass, N. et al., Acta Oncol 39 (2000): 931-934-   Marchi, S. et al., Cell Death. Dis. 3 (2012): e304-   Marioni, G. et al., Acta Otolaryngol. 129 (2009): 476-480-   Marnef, A. et al., Int. J Biochem. Cell Biol 41 (2009): 977-981-   Martin, L. et al., Oncogene 31 (2012): 4076-4084-   Mason, J. M. et al., Nucleic Acids Res. 43 (2015): 3180-3196-   Matsuda, R. et al., Br. J Cancer 104 (2011): 376-386-   Medina, P. P. et al., Epigenetics. 3 (2008): 64-68-   Mound, A. et al., Eur. J Cancer 49 (2013): 3738-3751-   Naidu, S. R. et al., Oncogene 28 (2009): 2492-2501-   Ng, Y. et al., J Biol Chem 279 (2004): 34156-34164-   Nibbe, R. K. et al., Mol Cell Proteomics. 8 (2009): 827-845-   Nishida, C. R. et al., Mol. Pharmacol. 78 (2010): 497-502-   O'Geen, H. et al., PLoS. Genet. 3 (2007): e89-   Ota, T. et al., Cancer Res 62 (2002): 5168-5177-   Paliouras, M. et al., Tumour. Biol 29 (2008): 63-75-   Papageorgio, C. et al., Int. J Oncol. 31 (2007): 1205-1211-   Papageorgis, P. et al., Cancer Res 70 (2010): 968-978-   Pohl, A. et al., Pharmacogenomics. J 11 (2011): 93-99-   Pollari, S. et al., Mol. Cancer Res 10 (2012): 597-604-   Qi, F. et al., Int. J Clin Exp. Pathol. 8 (2015): 1666-1673-   RefSeq, The NCBI handbook [Internet], Chapter 18, (2002),    http://www.ncbi.nlm.nih.gov/books/NBK21091/-   Reisman, D. N. et al., Cancer Res 63 (2003): 560-566-   Robles, L. D. et al., J Biol Chem 277 (2002): 25431-25438-   Ryu, B. et al., PLoS. One. 2 (2007): e594-   Sager, R. et al., Curr. Top. Microbiol. Immunol. 213 (Pt 1) (1996):    51-64-   Sakakura, C. et al., Anticancer Res 23 (2003): 3691-3697-   Sakurai, Y. et al., Mol. Pharm. 11 (2014): 2713-2719-   Sakurikar, N. et al., J Biol Chem 287 (2012): 39193-39204-   Sheng, S., Front Biosci. 9 (2004): 2733-2745-   Shibao, K. et al., Cell Calcium 48 (2010): 315-323-   Shu, G. S. et al., Cancer Biomark. 11 (2012): 107-114-   Stone, B. et al., Gene 267 (2001): 173-182-   Strekalova, E. et al., Clin. Cancer Res. (2015)-   Stutzer, I. et al., J Biol Chem 288 (2013): 10536-10547-   Su, K. C. et al., Dev. Cell 21 (2011): 1104-1115-   Sun, Y. et al., Oncotarget. 6 (2015b): 8244-8254-   Takashima, S. et al., Tumour. Biol. 35 (2014): 4257-4265-   Tatsuka, M. et al., Cancer Res 58 (1998): 4811-4816-   Tsui, K. H. et al., Sci. Rep. 5 (2015): 12870-   Van Ginkel, P. R. et al., Biochim. Biophys. Acta 1448 (1998):    290-297-   Vanaja, D. K. et al., Clin Cancer Res 12 (2006): 1128-1136-   Wang, G. et al., Oncogene 35 (2016): 651-661-   Wang, G. et al., Tumour. Biol 36 (2015a): 1055-1065-   Wang, Q. et al., PLoS. One. 8 (2013d): e70191-   Wang, W. et al., Int. J Cancer 124 (2009b): 521-530-   Wang, W. X. et al., Sichuan. Da. Xue. Xue. Bao. Yi. Xue. Ban. 40    (2009c): 857-860-   Wierinckx, A. et al., Endocr. Relat Cancer 14 (2007): 887-900-   Williams, K. A. et al., PLoS. Genet. 10 (2014): e1004809-   Wu, M. X., Apoptosis. 8 (2003): 11-18-   Wu, M. X. et al., Expert. Opin. Ther. Targets. 17 (2013): 593-606-   Wu, S. et al., Cell Cycle 13 (2014a): 2869-2878-   Wu, Z. et al., Neoplasia. 11 (2009): 66-76-   Xu, F. et al., Biochem. J 416 (2008): 15-26-   Yang, J. et al., Surg. Oncol 22 (2013): e53-e57-   Yang, L. et al., Cancer Res 71 (2011a): 5558-5568-   Yang, L. et al., Future. Oncol 8 (2012): 431-440-   Yang, X. et al., Biomed. Pharmacother. 67 (2013): 681-684-   Yang, Y. S. et al., Lung Cancer 74 (2011b): 12-24-   Yousef, G. M. et al., Tumour. Biol 26 (2005): 227-235-   Yu, B. et al., Exp. Cell Res 315 (2009): 3086-3098-   Zhang, F. et al., J Viral Hepat. 21 (2014a): 241-250-   Zhang, K. et al., Tumour. Biol 35 (2014e): 7669-7673-   Zhang, M. et al., Zhong. Nan. Da. Xue. Xue. Bao. Yi. Xue. Ban. 36    (2011a): 274-276-   Zhang, Y. et al., Cancer Sci. 101 (2010): 934-940-   Zheng, G. et al., Biochem. Biophys. Res Commun. 364 (2007): 344-350-   Follenzi A, et al. Nat Genet. 2000 June; 25(2):217-22.-   Zufferey R, et al. J Virol. 1999 April; 73(4):2886-92.-   Scholten K B, et al. Clin Immunol. 2006 May; 119(2):135-45.-   Gustafsson C, et al. Trends Biotechnol. 2004 July; 22(7):346-53.    Review.-   Kuball, J., et al. (2007). Blood 109, 2331-2338.-   Schmitt, T. M., et al. (2009). Hum. Gene Ther. 20, 1240-1248

1. A peptide consisting of the amino acid sequence of KLAVALLAA (SEQ IDNO: 210) in the form of a pharmaceutically acceptable salt.
 2. Thepeptide of claim 1, wherein the pharmaceutically acceptable salt is achloride salt, acetate salt, or trifluoro-acetate salt.
 3. Apharmaceutical composition comprising the peptide of claim 1 and animmune-stimulating adjuvant.
 4. The pharmaceutical composition of claim3, wherein the adjuvant is selected from the group consisting ofanti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 5. Thepharmaceutical composition of claim 15, wherein the adjuvant comprisesIL-2.
 6. The pharmaceutical composition of claim 15, wherein theadjuvant comprises IL-7.
 7. The pharmaceutical composition of claim 15,wherein the adjuvant comprises IL-15.
 8. The pharmaceutical compositionof claim 15, wherein the adjuvant comprises IL-21.
 9. A fusion proteincomprising a peptide consisting of the amino acid sequence of KLAVALLAA(SEQ ID NO: 210) and 80 N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii).
 10. A nucleic acid, encodingthe peptide of claim 1 linked to a heterologous promoter sequence. 11.An expression vector expressing the nucleic acid of claim
 10. 12. Arecombinant host cell comprising the peptide of claim 1, wherein saidhost cell is an antigen presenting cell, optionally a dendritic cell.13. A method for producing a peptide of claim 1, comprising culturingthe host cell that presents the peptide or expresses a nucleic acidencoding the peptide and isolating the peptide thereof from the hostcell or its culture medium.
 14. An in vitro method for producingactivated T lymphocytes, comprising contacting in vitro T cells withantigen loaded HLA-A*0201 expressed on the surface of anantigen-presenting cell for a period of time sufficient to activate saidT cells, wherein said antigen is a peptide consisting of the amino acidsequence of KLAVALLAA (SEQ ID NO: 210).
 15. The method of claim 14,wherein the T cells are autologous to the patient.
 16. The method ofclaim 14, wherein the T cells are obtained from a healthy donor.
 17. Themethod of claim 14, wherein the T cells are obtained from tumorinfiltrating lymphocytes or peripheral blood mononuclear cells.
 18. Themethod of claim 14, wherein the antigen presenting cell is infected withrecombinant virus expressing the peptide.
 19. The method of claim 18,wherein the antigen presenting cell is a dendritic cell or a macrophage.20. The method of claim 14, wherein the activated T cells compriseCD8-positive cells.